TW202302625A - Bioengineered immunomodulatory fusion protein compositions - Google Patents

Abstract

Provided herein, in some embodiments, are bioengineered immunomodulatory fusion proteins and uses thereof for modulating immune responses, as well as improving a response of a subject to a vaccine, or treating a disease or disorder, such as cancer or a pathogen infection.

Description

Immunomodulatory fusion protein composition modified by bioengineering

Provided herein, in some embodiments, are materials and methods for bioengineered immunomodulatory fusion proteins and their uses for detecting, modulating immune responses, and/or immune-related conditions; and for screening, diagnosing, Materials and methods for affecting, improving a subject's response to a pathogen or vaccine, or treating a disease or condition such as neoplasia, hyperplasia, cancer, or pathogen infection(s).

Immunity is a multifaceted host response that involves a variety of molecules. Manzoor Ahmad Mir, Chapter 1 - Introduction to Costimulation and Costimulatory Molecules, Editor(s): Manzoor Ahmad Mir, Developing Costimulatory Molecules for Immunotherapy of Diseases , Academic Press, 2015, Pages 1-43, ISBN 9780128025857. One such molecule involved in the immune response is CD40 ligand (CD40L) (CD154), a member of the tumor necrosis factor (TNF) superfamily that is present in various cell and tissue types and is highly expressed on CD4 ⁺ T cells on, and interacts with CD40 receptor polypeptides to enhance co-stimulatory signaling. CD40 receptor polypeptides are expressed on various innate and adaptive cells, including dendritic cells and B cells. Adv Drug Deliv Rev . 2019, 15;141:92-103; Immunol Rev . 2009 May; 229(1). CD40 agonism enhances antigen presentation and is useful for monoclonal antibody applications such as diagnostic, prognostic, and therapeutic ) of the appropriate target. Cancers (Basel) . 2021 Mar 15;13(6):1302.

The present inventors understood that, inter alia, CD40 agonist monoclonal antibodies performed poorly in the absence of Fc cross-links and exhibited little clinical benefit. In addition, systemic administration of CD40 agonist antibodies has been associated with adverse events and has been associated with hepatotoxicity, identifying and addressing an unmet need in the art such as improved engineered CD40 agonist Constructs of agents for use in modulating an immune response; and improving a subject's response to a vaccine, or treating a disease or condition, such as cancer or a pathogenic infection (eg, a viral infection). Thus, in one aspect, provided herein is a single chain trimeric CD40L Fc fusion protein comprising: (a) three CD40 ligand CD40L subunits covalently linked to each other by a peptide linker (CD40L trimeric body); and (b) Fc monomer peptide.

In some embodiments, the Fc monomer peptide is covalently linked to the CD40L trimer via a peptide tether. In some embodiments, the peptide tether comprises between 0 and 20 amino acids.

In some embodiments, the CD40 ligand subunits comprise a portion of the CD40L extracellular domain.

In some embodiments, the CD40L trimer is linked to the N-terminus of the Fc monomer peptide. In some embodiments, the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 1 to 12, or a fragment thereof.

In some embodiments, the CD40L trimer is linked to the C-terminus of the Fc monomer peptide. In some embodiments, the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 13 to 19, or a fragment thereof. In some embodiments, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO: 16 or a fragment thereof.

In some embodiments, the CD40 ligand subunits comprise any one of the sequences selected from SEQ ID NOS: 20-22, or a fragment thereof.

In some embodiments, the Fc monomeric peptide comprises human Fc sequences. In some embodiments, the human Fc sequence comprises a sequence selected from the group consisting of immunoglobulins IgG, IgA, IgM, IgD, and IgE. In some embodiments, the human Fc sequences comprise IgG sequences. In some embodiments, the IgG sequence is selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the IgG sequence comprises an IgG1 sequence. In some embodiments, the IgGl sequence comprises SEQ ID NO: 30 or 31, or a fragment thereof. In some embodiments, the IgG sequence comprises an IgG2 sequence. In some embodiments, the IgG2 sequence comprises SEQ ID NO: 29 or a fragment thereof.

In some embodiments, the peptide linker is selected from the group consisting of EGKSSGSGS (SEQ ID NO: 23) and (G ₃ S) ₃ (SEQ ID NO: 25).

In some embodiments, the peptide tether is selected from the group consisting of (G ₄ S) ₃ (SEQ ID NO: 24), (G ₄ S) ₂ (SEQ ID NO: 26), (G ₄ S) ₄ (SEQ ID NO:27), and the group consisting of G ₄ S (SEQ ID NO:28).

In some embodiments, the single chain trimeric CD40L Fc fusion protein enhances activation of the CD40 polypeptide compared to wild-type CD40L. In some embodiments, the activation of the CD40 polypeptide enhances the immunostimulatory function of dendritic cells, B cells, monocytes, and macrophages. In some embodiments, the activation of the CD40 polypeptide comprises enhanced T cell activation compared to wild-type CD40L. In some embodiments, the activation of the CD40 polypeptide comprises enhanced dendritic cell activation compared to wild-type CD40L. In some embodiments, the single-chain trimeric CD40L Fc fusion protein has enhanced anti-tumor activity compared to wild-type CD40L.

In another aspect, provided herein is a dimer comprising two single-chain trimeric CD40L Fc fusion proteins disclosed herein. In some embodiments, the dimerization system is a homodimer. In some embodiments, the dimerization system is formed by the association of the Fc monomeric peptides.

Accordingly, in yet another aspect, provided herein is a polynucleotide encoding a single-stranded trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits linked via a peptide linker covalently linked to each other; and (b) Fc monomeric peptides.

In yet another aspect, provided herein is a vector comprising a polynucleotide encoding a single-stranded trimeric CD40L fusion protein comprising: (a) three CD40L subunits expressed by a peptide the linkers are covalently linked to each other; and (b) the Fc monomer peptide.

In yet another aspect, provided herein is a host cell comprising a vector comprising a polynucleotide encoding a single-stranded trimeric CD40L fusion protein comprising: (a) three CD40L subunits units, which are covalently linked to each other by a peptide linker; and (b) an Fc monomer peptide.

In yet another aspect, provided herein is a host cell comprising a polynucleotide encoding a single-stranded trimeric CD40L fusion protein comprising: (a) three CD40L subunits in the form of are covalently linked to each other by a peptide linker; and (b) an Fc monomer peptide.

In yet another aspect, provided herein is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a single-chain trimeric CD40L fusion protein comprising: (a) three CD40L subunits formed by peptide the linkers are covalently linked to each other; and (b) the Fc monomer peptide.

In yet another aspect, provided herein is a kit comprising a single-chain trimeric CD40L fusion protein comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and (b) Fc monomeric peptides.

In one aspect, provided herein is a system comprising a building block for providing a single-chain trimeric CD40L fusion protein comprising: (a) three CD40L subunits interconnected by a peptide linker covalently linked; and (b) an Fc monomer peptide. In another aspect, provided herein is a system comprising a building block for providing a dimer comprising two single-chain trimeric CD40L Fc fusion proteins each comprising: (a) Three CD40L subunits covalently linked to each other by a peptide linker; and (b) Fc monomer peptide.

In one aspect, provided herein is a method of activating a CD40 polypeptide comprising contacting the CD40 polypeptide with a single chain trimeric CD40L Fc fusion protein disclosed herein, wherein the single chain trimeric CD40L Fc fusion protein is activated upon binding The CD40 polypeptide. In another aspect, provided herein is a method of activating a CD40 polypeptide comprising contacting the CD40 polypeptide with a dimer comprising two single chain trimeric CD40L Fc fusion proteins disclosed herein, wherein the The single-chain trimeric CD40L Fc fusion protein dimer activates the CD40 polypeptide upon binding. In yet another aspect, provided herein is a method of activating a T cell comprising contacting the T cell with an antigen-presenting cell in the presence of a single-chain trimeric CD40L Fc fusion protein disclosed herein, wherein the antigen-presenting cell expresses CD40 polypeptide, and wherein the single-chain trimeric CD40L Fc fusion protein activates the T cell when binding to the CD40 polypeptide. In yet another aspect, provided herein is a method of activating a T cell comprising contacting the T cell with an antigen presenting cell in the presence of a dimer comprising two single-chain trimers disclosed herein. A CD40L Fc fusion protein, wherein the antigen presenting cell expresses a CD40 polypeptide, and wherein the single chain trimeric CD40L Fc fusion protein dimer activates the T cell upon binding the CD40 polypeptide. In some embodiments, the antigen presenting cell presents antigen to the T cell. In yet another aspect, provided herein is a method of activating dendritic cells comprising contacting the CD40 polypeptide with a single-chain trimeric CD40L Fc fusion protein disclosed herein, wherein the single-chain trimeric CD40L Fc fusion protein is in The dendritic cells are activated upon binding of the CD40 polypeptide. In yet another aspect, provided herein is a method of activating dendritic cells comprising contacting the CD40 polypeptide with a dimer comprising two single chain trimeric CD40L Fc fusion proteins disclosed herein, Wherein the single-chain trimeric CD40L Fc fusion protein dimer activates the dendritic cell when combined with the CD40 polypeptide.

In some embodiments, the method is performed in vitro. In some embodiments, the method is performed in vivo. In some embodiments, the contacting further comprises administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the single-chain trimeric CD40L Fc fusion protein. In some embodiments, the contacting enhances an innate anti-neoplastic immune response.

In one aspect, provided herein is a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a single-chain trimeric CD40L Fc fusion protein disclosed herein. In another aspect, provided herein is a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a dimer comprising two single chain trimeric CD40L Fc fusions disclosed herein protein.

In some embodiments, the method further comprises administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the single-chain trimeric CD40L Fc fusion protein. In some embodiments, the treatment enhances an innate anti-neoplastic immune response. In some embodiments, the method further comprises co-administration of a second therapy. In some embodiments, the cancer is selected from the group consisting of melanoma, mesothelioma, advanced solid tumors, and lymphoma.

In one aspect, provided herein is a method for producing a single-stranded trimeric CD40L Fc fusion protein or fragment thereof, comprising: (a) introducing into a host cell a polynucleotide encoding a single-stranded CD40L Fc fusion protein disclosed herein; chain trimeric CD40L Fc fusion protein; (b) culturing the host cell under conditions to produce the single chain trimeric CD40L Fc fusion protein or a fragment thereof; and (c) recovering the single chain trimeric CD40L Fc fusion protein from the cell or culture CD40L Fc fusion protein or fragment thereof. In another aspect, provided herein is a method of producing a dimer comprising two single-stranded trimeric CD40L Fc fusion proteins, the method each comprising: (a) introducing a polynucleotide into a host cell, the A polynucleotide encoding a single-chain trimeric CD40L Fc fusion protein disclosed herein; (b) culturing the host cell under conditions to produce the single-chain trimeric CD40L Fc fusion protein or a fragment thereof; (c) from the cell or recovering the single-chain trimeric CD40L Fc fusion protein or fragment thereof from culture; and (d) combining the single-chain trimeric CD40L Fc fusion protein or fragment thereof under conditions favorable for dimerization. In yet another aspect, provided herein is a method of producing a pharmaceutical composition having a single-chain trimeric CD40L Fc fusion protein or fragment thereof comprising combining a single-chain trimeric CD40L Fc fusion protein disclosed herein or a fragment thereof with A pharmaceutically acceptable carrier is combined to obtain the pharmaceutical composition.

In one aspect, provided herein is a polypeptide comprising a single-chain trimeric CD40L fusion protein, wherein the single-chain trimeric CD40L fusion protein comprises three CD40L subunits connected to each other by a peptide linker covalently linked. In some embodiments, the CD40L subunits comprise a portion of the CD40L extracellular domain. In some embodiments, the CD40L subunits comprise any one of the sequences selected from SEQ ID NOS: 20-22, or a fragment thereof.

In some embodiments, at least one of the peptide linkers is selected from the group consisting of EGKSSGSGS (SEQ ID NO: 23) and (G ₃ S) ₃ (SEQ ID NO: 25). In some embodiments, at least two of the peptide linkers are selected from the group consisting of EGKSSGSGS (SEQ ID NO: 23) and (G ₃ S) ₃ (SEQ ID NO: 25). In some embodiments, at least two of the peptide linkers have the same sequence.

In some embodiments, the single-chain trimeric CD40L fusion protein comprises any one sequence selected from SEQ ID NO: 35 to 38, or a fragment thereof. In some embodiments, the single chain trimeric CD40L fusion protein is fused to a peptide or polypeptide not derived from CD40L.

In another aspect, the single chain trimeric CD40L fusion protein is fused to a peptide tether. In some embodiments, the peptide tether is selected from the group consisting of (G ₄ S) ₃ (SEQ ID NO: 24), (G ₄ S) ₂ (SEQ ID NO: 26), (G ₄ S) ₄ (SEQ ID NO:27), and the group consisting of G ₄ S (SEQ ID NO:28). In some embodiments, the peptide tether is fused to the N-terminus of the single-chain trimeric CD40L fusion protein. In some embodiments, the peptide tether is fused to the C-terminus of the single-chain trimeric CD40L fusion protein.

In another aspect, the single chain trimeric CD40L fusion protein is fused to an Fc monomeric peptide. In some embodiments, the Fc monomeric peptide comprises human Fc sequences. In some embodiments, the human Fc sequence comprises a sequence selected from the group consisting of immunoglobulins IgG, IgA, IgM, IgD, and IgE. In some embodiments, the human Fc sequences comprise IgG sequences. In some embodiments, the IgG sequence is selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the IgG sequence comprises an IgG1 sequence. In some embodiments, the IgGl sequence comprises SEQ ID NO: 30 or 31, or a fragment thereof. In some embodiments, the IgG sequence comprises an IgG2 sequence. In some embodiments, the IgG2 sequence comprises SEQ ID NO: 29 or a fragment thereof.

In some embodiments, the single chain trimeric CD40L fusion protein is fused to the Fc monomeric peptide via a peptide tether. In some embodiments, the peptide tether comprises between 0 and 20 amino acids. In some embodiments, the peptide tether is selected from the group consisting of (G ₄ S) ₃ (SEQ ID NO: 24), (G ₄ S) ₂ (SEQ ID NO: 26), (G ₄ S) ₄ (SEQ ID NO:27), and the group consisting of G ₄ S (SEQ ID NO:28).

In some embodiments, the CD40L trimer is linked to the N-terminus of the Fc monomer peptide. In some embodiments, the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 1 to 12, or a fragment thereof. In some embodiments, the CD40L trimer is linked to the C-terminus of the Fc monomer peptide. In some embodiments, the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 13 to 19, or a fragment thereof.

In another aspect, the single-chain trimeric CD40L fusion protein enhances activation of the CD40 polypeptide compared to wild-type CD40L. In some embodiments, the activation of the CD40 polypeptide enhances the immunostimulatory function of T cells and/or B cells. In some embodiments, the activation of the CD40 polypeptide comprises B cells, CD4+ T cells, CD8+ T cells, dendritic cells, macrophages, natural killer cells, monocytes, granules compared to wild-type CD40L Enhanced activation of globules, eosinophils, and/or neutrophils.

In some embodiments, the activation of the CD40 polypeptide comprises increasing expression of the CD40 polypeptide. In some embodiments, the single-chain trimeric CD40L fusion protein has enhanced anti-tumor activity compared to wild-type CD40L. In some embodiments, the single-chain trimeric CD40L fusion protein has enhanced pro-inflammatory activity compared to wild-type CD40L. In some embodiments, the single-chain trimeric CD40L fusion protein enhances clearance of infectious pathogens compared to wild-type CD40L.

In another aspect, the single chain trimeric CD40L fusion protein increases antibody production by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55% of a B cell population , about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%.

In another aspect, the single-chain trimeric CD40L fusion protein increases the secretion of pro-inflammatory cytokines in a population of T cells. In some embodiments, the pro-inflammatory cytokines are IL-1, IL-2, IL-6, IL 12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN- γ, or any combination thereof. In some embodiments, the cytokine production is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300% , about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%.

In another aspect, the single-chain trimeric CD40L fusion protein increases the minimal percentage of phagocytic macrophages in a population of macrophages to about 10%, about 20%, about 30%, about 40%, about 45%, About 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

In some embodiments, the single chain trimeric CD40L fusion protein increases the minimal percentage of antigen presenting dendritic cells in a population of dendritic cells to about 10%, about 20%, about 30%, about 40%, about 45%, About 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

In another aspect, the polypeptide is conjugated to an agent. In some embodiments, the agent is selected from the group consisting of radioisotopes, metal chelators, enzymes, fluorescent compounds, bioluminescent compounds, or chemiluminescent compounds.

In another aspect, provided herein is a polynucleotide encoding a single-stranded trimeric CD40L fusion protein, wherein the single-stranded trimeric CD40L fusion protein comprises three CD40L subunits derived from are covalently linked to each other by peptide linkers.

In another aspect, provided herein is a vector comprising a polynucleotide encoding a single-stranded trimeric CD40L fusion protein, wherein the single-stranded trimeric CD40L fusion protein comprises three CD40L subunits, the The CD40L subunits are covalently linked to each other by peptide linkers.

In another aspect, provided herein is a host cell comprising a vector comprising a polynucleotide encoding a single-stranded trimeric CD40L fusion protein, wherein the single-stranded trimeric CD40L fusion protein comprises three CD40L subunits covalently linked to each other via a peptide linker.

In another aspect, provided herein is a host cell comprising a polynucleotide encoding a single-stranded trimeric CD40L fusion protein, wherein the single-stranded trimeric CD40L fusion protein comprises three CD40L subunits, The CD40L subunits are covalently linked to each other by a peptide linker.

In another aspect, provided herein is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polypeptide comprising a single-chain trimeric CD40L fusion protein, wherein the single-chain trimeric CD40L fusion protein comprises three CD40L subunits covalently linked to each other via a peptide linker.

In another aspect, provided herein is a kit comprising polypeptides comprising a single-chain trimeric CD40L fusion protein, wherein the single-chain trimeric CD40L fusion protein comprises three CD40L subunits, the CD40L subunits The units are covalently linked to each other via peptide linkers.

In another aspect, provided herein is a system comprising a means for providing a polypeptide comprising a single-chain trimeric CD40L fusion protein, wherein the single-chain trimeric CD40L fusion protein comprises three CD40L subunits unit, the CD40L subunits are covalently linked to each other by a peptide linker.

In another aspect, provided herein is a method for activating a target cell expressing a CD40 polypeptide comprising contacting the target cell with an effective amount of a polypeptide comprising a single-chain trimeric A CD40L fusion protein, wherein the single-chain trimeric CD40L fusion protein comprises three CD40L subunits covalently linked to each other by a peptide linker, wherein the single-chain trimeric CD40L fusion protein binds the CD40 polypeptide activate the target cells.

In some embodiments, activation of the target cell is measured as increased proliferation or maturation of the target cell. In some embodiments, the proliferation or maturation of the target cell is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% , about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%.

In some embodiments, activation of the target cell is measured as an extended survival time of the target cell. In some embodiments, the survival time of the target cells is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, About 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300% %, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%.

In some embodiments, the target cell is an antigen presenting cell. In some embodiments, the target cells are natural killer cells, B cells, dendritic cells, macrophages, monocytes, granulocytes, eosinophils, neutrophils, or combinations thereof.

In another aspect, provided herein is a method for promoting antibody production in a population of B cells comprising contacting the B cells with an effective amount of a polypeptide comprising a single-chain three A polyCD40L fusion protein, wherein the single chain trimeric CD40L fusion protein comprises three CD40L subunits covalently linked to each other by a peptide linker.

In some embodiments, antibody production by the B cell population is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% , about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%.

In another aspect, the B cells are contacted with a polypeptide, or a polynucleotide encoding the polypeptide, comprising a single-chain trimeric CD40L fusion protein, in the presence of an antigen or a polynucleotide encoding the antigen, wherein the The single chain trimeric CD40L fusion protein comprises three CD40L subunits covalently linked to each other by a peptide linker, and wherein the antibody produced by the B cells specifically binds to the antigen. In some embodiments, the method further promotes the formation of memory B cells capable of producing the antibody in response to the antigen.

In some embodiments, the polypeptide or the polynucleotide is in a vaccine composition or an adjuvant composition. In some embodiments, the antigen or polynucleotide encoding the antigen is in a vaccine composition. In some embodiments, the antigen is derived from or derived from an infectious pathogen. In some embodiments, the infectious agent is a virus, bacterium, fungus, parasite, or a combination thereof. In some embodiments, the antigen is derived from or derived from a diseased cell. In some embodiments, the diseased cell is a cancer cell. In some embodiments, the diseased cell line is a cell infected with an infectious pathogen. In some embodiments, the infectious agent is a virus, bacterium, fungus, parasite, or a combination thereof.

In some embodiments, the antigen is presented by an antigen presenting cell. In some embodiments, the antigen presenting cell line is dendritic cells. In some embodiments, the antigen is associated with an MHC class I complex or an MHC class II complex.

In another aspect, provided herein is a method of increasing the secretion of pro-inflammatory cytokines in a population of T cells, comprising presenting the population of T cells with an antigen in the presence of an effective amount of a polypeptide or a polynucleotide encoding the polypeptide. The cell population is contacted with the polypeptide comprising a single-chain trimeric CD40L fusion protein, wherein the single-chain trimeric CD40L fusion protein comprises three CD40L subunits covalently linked to each other by a peptide linker. In some embodiments, the cytokine is IL-1, IL-2, IL-6, IL 12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, or any combination thereof. In some embodiments, the cytokine production is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300% , about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%.

In another aspect, provided herein is a method of increasing phagocytosis of diseased cells by a population of macrophages, comprising causing the diseased cells, the macrophages, or both the diseased cells and the macrophages to contacting an effective amount of a polypeptide comprising a single-chain trimeric CD40L fusion protein, wherein the single-chain trimeric CD40L fusion protein comprises three CD40L subunits, the CD40L subunits being formed by Peptide linkers are covalently linked to each other.

In some embodiments, the minimum percentage of phagocytic macrophages in the population of macrophages is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, About 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the phagocytosis of macrophages is measured by co-culturing macrophages labeled with a first fluorescent dye with diseased cells labeled with a second fluorescent dye, wherein the first The fluorescent dye is different from the second fluorescent dye. In some embodiments, the percentage of phagocytic macrophages is measured by determining the percentage of macrophages comprising the diseased cells. In some embodiments, the diseased cells are cancer cells or cells infected with infectious pathogens. In some embodiments, the infectious agent is a virus, bacterium, fungus, parasite, or a combination thereof.

In another aspect, provided herein is a method of increasing antigen presentation by a population of dendritic cells comprising contacting the dendritic cells in the presence of the antigen with an effective amount of a polypeptide or a polynucleotide encoding the polypeptide, the The polypeptide comprises a single-chain trimeric CD40L fusion protein, wherein the single-chain trimeric CD40L fusion protein comprises three CD40L subunits covalently linked to each other by a peptide linker.

In some embodiments, the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55% %, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the antigen presentation by the dendritic cells is measured by co-culturing dendritic cells labeled with a first fluorescent dye with the antigen labeled with a second fluorescent dye, wherein The first fluorescent dye is different from the second fluorescent dye. In some embodiments, the percentage of dendritic cells presenting the antigen is measured by determining the percentage of dendritic cells in the population of dendritic cells that co-localize with the antigen.

In some embodiments, the antigen is derived from or derived from an infectious pathogen. In some embodiments, the infectious agent is a virus, bacterium, fungus, parasite, or a combination thereof. In some embodiments, the antigen is derived from or derived from a diseased cell. In some embodiments, the diseased cell is a cancer cell. In some embodiments, the diseased cell line is a cell infected with an infectious pathogen. In some embodiments, the infectious agent is a virus, bacterium, fungus, parasite, or a combination thereof.

In some embodiments, the polypeptide or the polynucleotide is in a vaccine composition or an adjuvant composition. In some embodiments, the antigen or polynucleotide encoding the antigen is in a vaccine composition.

In another aspect, provided herein is a method of increasing expression of a CD40 polypeptide in a target cell comprising contacting the target cell with an effective amount of a polypeptide comprising a single-chain trimeric CD40L fusion Protein, wherein the single-chain trimeric CD40L fusion protein comprises three CD40L subunits, and the CD40L subunits are covalently linked to each other by a peptide linker.

In some embodiments, the target cell is a diseased cell. In some embodiments, the diseased cell is a cancer cell. In some embodiments, the diseased cell line is a cell infected with an infectious pathogen. In some embodiments, the infectious agent is a virus, bacterium, fungus, parasite, or a combination thereof.

In some embodiments, the target cell is an immune cell. In some embodiments, the target cell is an antigen presenting cell. In some embodiments, the target cells are natural killer cells, B cells, dendritic cells, macrophages, monocytes, granulocytes, eosinophils, neutrophils, or combinations thereof.

In some embodiments, the diseased cell population is reduced by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70% %, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

In another aspect, provided herein is a method of creating a pro-inflammatory environment in tissue surrounding a population of diseased cells, comprising contacting the tissue with an effective amount of a polypeptide comprising a single-chain three A polyCD40L fusion protein, wherein the single chain trimeric CD40L fusion protein comprises three CD40L subunits covalently linked to each other by a peptide linker.

In some embodiments, the activated B cells, CD4+ T cells, CD8+ T cells, dendritic cells, macrophages, natural killer cells, monocytes, granulocytes, eosinophils, and/or activated B cells in the tissue Increased infiltration of neutral spheres.

In some embodiments, the concentration of pro-inflammatory cytokines is increased in the tissue. In some embodiments, the pro-inflammatory cytokines are IL-1, IL-2, IL-6, IL 12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN- γ, or any combination thereof.

In some embodiments, antigen presentation by antigen presenting cells from or derived from the diseased cells is increased in the tissue.

In some embodiments, phagocytosis of the diseased cells is increased in the tissue.

In some embodiments, apoptosis of the diseased cells caused by cell-mediated cytotoxicity is increased in the tissue. In some embodiments, apoptosis of the diseased cells caused by antibody-dependent cellular cytotoxicity is increased in the tissue.

In some embodiments, the diseased cell population is reduced in the tissue. In some embodiments, the diseased cell population is reduced by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% in the tissue %, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

In some embodiments, the method is performed in vitro or in vivo.

In another aspect, provided herein is a method for eliminating diseased cells in a subject, comprising administering an effective amount of a polypeptide comprising a single-chain trimeric CD40L fusion protein, wherein the single-chain Trimeric CD40L fusion proteins comprise three CD40L subunits covalently linked to each other by a peptide linker.

In some embodiments, the diseased cell does not express a CD40 polypeptide. In some embodiments, the diseased cell expresses a CD40 polypeptide. In some embodiments, the diseased cell is a cancer cell. In some embodiments, the diseased cell line is a cell infected with an infectious pathogen. In some embodiments, the infectious agent is a virus, bacterium, fungus, parasite, or a combination thereof.

In another aspect, provided herein is a method of treating cancer in a subject in need thereof, comprising administering an effective amount of a polypeptide comprising a single-chain trimeric CD40L fusion protein, or a polynucleotide encoding the polypeptide, wherein the The single-chain trimeric CD40L fusion protein comprises three CD40L subunits covalently linked to each other by a peptide linker. In some embodiments, the treatment enhances an innate, humoral, or cell-mediated anti-tumor immune response. In some embodiments, the method further comprises co-administration of a second therapy. In some embodiments, the cancer is selected from the group consisting of melanoma, mesothelioma, advanced solid tumors, and lymphoma.

In another aspect, provided herein is a method of treating an infection in a subject in need thereof, comprising administering an effective amount of a polypeptide comprising a single-chain trimeric CD40L fusion protein, or a polynucleotide encoding the polypeptide, wherein the The single-chain trimeric CD40L fusion protein comprises three CD40L subunits covalently linked to each other by a peptide linker. In some embodiments, the treatment enhances the innate, humoral, or cell-mediated immune response against infection.

In some embodiments, the polypeptide or the polynucleotide is co-administered with a vaccine composition for preventing the infection in the subject. In some embodiments, the polypeptide or the polynucleotide is co-administered simultaneously or sequentially with the vaccine composition.

Cross-reference to related applications

This application claims priority to: U.S. Serial No. 63/160,686, filed March 12, 2021; U.S. Serial No. 63/160,688, filed March 12, 2021; U.S. Serial No. 63/160,691, filed March 12; U.S. Serial No. 63/160,693, filed March 12, 2021; U.S. Serial No. 63, filed March 12, 2021 /160,694, each of which is incorporated herein by reference in its entirety.

The present disclosure is based in part on the surprising discovery of a single CD40L Fc fusion protein with favorable biophysical properties and enhanced CD40 activation, which leads to enhanced T cell activation and dendritic cell activation. Accordingly, the compositions and methods of the invention provide means for novel and improved therapeutic strategies targeting the CD40-CD40L pathway.

Individual publications, papers, and patents have been cited or described in the prior art and throughout the specification; each of these references is hereby incorporated by reference in its entirety. The discussion of documents, acts, materials, devices, articles, or the like in this specification is included for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any disclosed or claimed invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical fields to which this invention pertains. In other respects, certain terms used herein have the meanings as set forth in this specification.

It must be noted that the singular forms "one (a/an)" and "the (the)" used herein and in the appended claims include plural reference unless the context clearly states otherwise.

Unless otherwise indicated, any numerical values, such as concentrations or concentration ranges, recited herein are to be understood as being modified in all instances by the word "about". Accordingly, numerical values generally include ± 10% of the stated value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL to 1.1 mg/mL. Likewise, a concentration range of 1% to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, expressly used numerical ranges include all possible subranges, all individual values within that range, including integers and fractions of values within such ranges, unless the context clearly dictates otherwise.

Unless otherwise indicated, the word "at least" preceding a series of elements should be understood as referring to each element in the series.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by this invention.

As used herein, the terms "comprises, comprising", "includes, including", "has, having", "contains or containing" or any other variation thereof shall is understood to implicitly include said whole or group of wholes, but not to exclude any other whole or group of wholes, and is intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or device comprising a listed element is not necessarily limited to only those elements, but may include a composition, mixture, process, method, article, or other components inherent in the device. Furthermore, unless expressly stated to the contrary, "or (or)" means an inclusive "or" rather than an exclusive "or". For example, any of the following satisfies the condition A or B: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and A and B Both are true (or exist).

As used herein, the linking term "and/or" between a plurality of stated elements is understood to encompass both individual and combined options. For example, where two elements are joined by "and/or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element in the absence of the first element. The third option refers to the applicability of the first element and the second element together. Either of these options should be understood to fall within that meaning, and thus satisfy the requirements of the term "and/or" as used herein. The concurrent applicability of more than one of these options is also to be understood as falling within this meaning, and thus fulfills the requirement of the word "and/or".

As used herein, the term "consists of", or variations such as "consist of or consisting of" (as used throughout the specification and claims) indicates that Any reference to a whole or group of wholes, but no additional whole or group of wholes may be added to a given method, structure, or composition.

As used herein, the phrase "consists essentially of" or variations such as "consist essentially of or consisting essentially of" (as throughout the specification and claims As used in ) designation includes any recited whole or group of wholes, and may optionally include any recited whole or group of wholes that does not substantially alter the basic or novel properties of a specified method, structure, or composition. See M.P.E.P. § 2111.03.

"Subject" as used herein means any animal, preferably a mammal, most preferably a human. The term "mammal" as used herein encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, and the like. In a specific embodiment, the subject is a human being. In some embodiments, a subject is a subject in need.

As used herein, the term "treat/treatment/treating" refers to any method for the partial or complete alleviation, improvement or improvement of one or more symptoms or characteristics of a particular disease, disorder, and/or condition , alleviate, inhibit, prevent, delay onset, reduce severity, and/or reduce incidence. To reduce the risk of developing disease-related lesions, treatment can be administered to subjects who exhibit no signs of disease and/or who exhibit only early signs of disease.

It should also be understood that the terms "about", "approximately", "generally", "substantially" and similar terms used herein are intended to refer to groups of preferred inventions. When specifying dimensions or features, it is indicated that the dimensions/features are not strict boundaries or parameters, and minor variations that will be functionally the same or similar to them as understood by those skilled in the art are not excluded. At a minimum, such references to numerical parameters include variations subject to the use of mathematical and industrial principles accepted in the art (e.g., rounding, errors of measurement or other systems, manufacturing tolerances, etc. ), will not change at least significant digits.

The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences means when compared and aligned for maximum correspondence, such as using the following sequence comparison Two or more sequences or subsequences are identical, or have a specified percentage of identical amino acid residues or nucleotides, as measured by one of an algorithm or by visual inspection.

For sequence comparison, typically one sequence is treated as a reference sequence, and test sequences are compared to it. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated (if necessary), and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequence(s) relative to the reference sequence, based on the specified program parameters.

Optimal alignment of sequence comparisons can be performed by, for example, the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981); Needleman & Wunsch, J. Mol. Biol. 48:443 (1970) homology comparison algorithm; Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988) similarity search method; these algorithms (Genetics Computer Group , 575 Science Dr., Madison, WI, computerized implementation of GAP, BESTFIT, FASTA, and TFASTA) in the Wisconsin Genetics Software Package; visual inspection (see generally Current Protocols in Molecular Biology, FM Ausubel et al. , eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms suitable for use in determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. al. (1997) Nucleic Acids Res. 25: 3389-3402. Software for performing BLAST analyzes is made available to the public by the National Center for Biotechnology Information. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which match when aligned with words of the same length in the database sequence Or satisfy some positive-valued threshold score T. T refers to the neighborhood word score threshold (Altschul et al. , supra). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing them. Next, word hits are extended in both directions along each sequence to increase the degree to which the cumulative alignment score can be increased.

For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for pairs of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate cumulative scores. Extension of word hits in each direction stops when: the cumulative alignment score falls by the amount X from its maximum achievement value; the cumulative score becomes zero or below due to the accumulation of one or more negative-scoring residue alignments; or when at the end of any sequence. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses the following as defaults: wordlength (W) of 11, expectation (E) of 10, M=5, N=-4, and comparison of two strands. For amino acid sequences, the BLASTP program uses the following as defaults: word length (W) 3, expectation (E) 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993) ). One such measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in the comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid and the polypeptide encoded by the second nucleic acid have immunological cross-reactivity as described below. Thus, a polypeptide is generally substantially identical to a second polypeptide, for example, when two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

As used herein, the term "polynucleotide (polynucleotide)" is synonymously referred to as "nucleic acid molecule (nucleic acid molecule)", "nucleotide (nucleotide)", or "nucleic acid (nucleic acid)", and refers to any Polyribonucleotides or polydeoxyribonucleotides, which may be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions , a hybrid molecule comprising DNA and RNA which may be single-stranded or (more typically) double-stranded or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases and DNA or RNA having a backbone modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA; thus, "polynucleotide" includes chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as DNA and RNA characteristic of viruses and cells chemical form. "Polynucleotide" also includes relatively short strands of nucleic acid, commonly referred to as oligonucleotides.

The term "encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide (such as a gene, cDNA, or mRNA) to serve as a template for the synthesis of other polymers and macromolecules in biological processes, such polymers and Macromolecules have a defined nucleotide sequence (eg, rRNA, tRNA, and mRNA) or a defined amino acid sequence, and biological properties derived therefrom. Thus, a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence, and which is generally provided in the Sequence Listing, and the non-coding strand, which serves as a template for transcription of a gene or cDNA, may both be referred to as the encoded protein or other product of the gene or cDNA.

Unless otherwise indicated, "nucleotide sequence encoding an amino acid sequence" (or equivalent phrase) includes all nucleic acid sequences that are degenerate versions of each other and encode the same amino acid sequence nucleotide sequence. A phrase nucleotide sequence encoding a protein or RNA may also include introns (provided that the nucleotide sequence encoding the protein may, in some versions, contain one or more introns).

As used herein, the term "vector" is a replicon into which another nucleic acid segment is operably inserted, thereby allowing the segment to be replicated or expressed.

As used herein, the term "host cell" refers to a cell comprising a nucleic acid molecule of the invention. A "host cell" can be any type of cell, such as a primary cell, a cell in culture, or a cell from a cell line. In one embodiment, a "host cell" is a cell transfected with a nucleic acid molecule disclosed herein. In another embodiment, a "host cell" is the progeny or potential progeny of such transfected cells. Progeny of a cell may or may not be identical to the parent cell due to, for example, mutations or environmental influences that may occur in subsequent generations or when the nucleic acid molecule integrates into the host cell genome.

As used herein, the term "expression" refers to the biosynthesis of a gene product. The term encompasses the transcription of a gene into RNA. The term also encompasses translation of RNA into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications. The expressed molecule can be within the cytoplasm of the host cell, enter the extracellular environment (such as the growth medium of a cell culture), or be anchored to the cell membrane.

As used herein, the term "peptide", "polypeptide", or "protein" may refer to a molecule comprising amino acids and may be recognized as a protein by those of ordinary skill in the art. The conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "peptide", "polypeptide", and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer can be linear or branched, it can contain modified amino acids, and it can be non-amino acid interrupted. The term also encompasses amino acid polymers that have been modified naturally or by interference; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as with labeling components join. Also included within this definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

Peptide sequences described herein are written according to the usual convention, with the N-terminal region of the peptide written on the left and the C-terminal region on the right. Although amino acids are known to have isomeric forms, unless expressly indicated otherwise, they are expressed as L-form amino acids.

As used herein, the terms "antibody" or "immunoglobulin" are used in a broad sense and include human, humanized, complex, and chimeric antibodies and antibody fragments, which are Single or multiple plants. Broadly, an antibody is a protein or peptide chain that exhibits binding specificity for a particular antigen. Naturally occurring antibodies are Y-shaped molecules composed of two heavy chains and two light chains that fold into constant and variable domains. The structure of antibodies, including heavy and light chains and the constant and variable regions within each of these heavy and light chains, is well known. That is, in addition to the heavy and light chain constant domains, an antibody contains an antigen-binding region consisting of a light chain variable region (VL) and a heavy chain variable region (VH), which Each of the chain variable regions contains three domains, complementarity determining region 1 (CDR1), CDR2, and CDR3. "CDR" means one of the three hypervariable regions (HCDR1, HCDR2, or HCDR3) within the non-framework region of the VH β-sheet framework of an immunoglobulin (Ig or antibody), or the VL β-sheet framework of an antibody One of the three hypervariable regions (LCDR1, LCDR2, or LCDR3) within the non-architectural region. Immunoglobulins can be divided into five major classes (ie, IgA, IgD, IgE, IgG, and IgM), depending on the amino acid sequence of the heavy chain constant domain. The IgA and IgG lines are further subdivided into isotypes IgAl, IgA2, IgGl, IgG2, IgG3, and IgG4.

The term "constant region" or "constant domain" refers to the carboxy-terminal portion of the light and heavy chains, which are not directly involved in antibody binding to antigen, but which exhibit various effector functions (such as binding to Fc receptors). body interactions). These terms refer to the portion of the immunoglobulin molecule that has a more conserved amino acid sequence relative to other portions of the immunoglobulin, including the variable region containing the antigen binding site. The constant region may contain the CH1, CH2, and CH3 regions of the heavy chain and the CL region of the light chain.

The term "Fc region" refers to the carboxy-terminal portion of the constant region of an antibody, and it encompasses the CH2 and CH3 regions of the heavy chain. The two CH3 domains interact with each other to form a homodimer, which in turn leads to Fc dimerization.

As used herein, the term "Fc monomer" or "mFc" or "Fc monomer peptide" refers to a molecule that includes the carboxy-terminal portion of a monomeric Fc region that is It consists of the CH2 region and CH3 region of the heavy chain or part thereof. In some embodiments, the Fc monomeric peptide comprises human Fc sequences. In some embodiments, the human Fc sequence comprises a sequence selected from the group consisting of immunoglobulins IgG, IgA, IgM, IgD, and IgE. In one embodiment, the human Fc sequences comprise IgG sequences. In some embodiments, the IgG sequence is selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the IgG sequence is an IgG1 antibody. In some embodiments, the IgGl sequence comprises SEQ ID NO: 30 or 31, or a fragment thereof. In one embodiment, the IgGl sequence comprises SEQ ID NO: 30 or a fragment thereof. In another embodiment, the IgGl sequence comprises SEQ ID NO: 31 or a fragment thereof. In some embodiments, the IgG sequence comprises an IgG2 sequence. In some embodiments, the IgG2 sequence comprises SEQ ID NO: 29 or a fragment thereof.

The single-chain trimeric CD40L Fc fusion proteins provided herein may comprise Fc monomeric peptides of any of the five major classes or corresponding subclasses. In specific embodiments, the Fc monomeric peptides provided herein are IgGl, IgG2, IgG3, or IgG4. In further embodiments, the Fc monomeric peptides provided herein are human IgG1 isotype and human IgG2 isotype. In additional embodiments, the Fc monomer peptide component of the single chain trimeric CD40L Fc fusion protein has silencing effector function.

Thus, the single chain trimeric CD40L Fc fusion proteins provided herein can contain Fc monomeric peptides corresponding to the constant domains of the kappa or lambda light chains. According to specific embodiments, the single chain trimeric CD40L Fc fusion proteins disclosed herein comprise Fc monomers with heavy and/or light chain constant regions from rat or human antibodies.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibody lines that make up the population except for possible naturally occurring mutations that may be present in minor amounts identical. The monoclonal antibodies disclosed herein can be produced by fusionoma method, phage display technology, single lymphocyte gene selection technology, or by recombinant DNA method. For example, a monoclonal antibody can be produced by a fusion tumor comprising B cells obtained from a transgenic non-human animal, such as a transgenic mouse or rat, with a transgene comprising a human heavy chain. and the gene body of the light chain transgene. Thus, the single chain trimeric CD40L Fc fusion proteins provided herein can comprise Fc monomers from monoclonal antibodies.

The term "single-chain antibody" as used herein refers to a single-chain antibody known in the art, which comprises a variable heavy chain connected by a short peptide of about 15 to about 20 amino acids. region and light chain variable region. Thus, the single chain trimeric CD40L Fc fusion proteins provided herein can comprise Fc monomers from single chain antibodies.

As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human produced using any technique known in the art. This definition of human antibody includes whole or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide. Thus, the single chain trimeric CD40L Fc fusion proteins provided herein can comprise Fc monomers from human antibodies.

As used herein, the term "humanized antibody" refers to a non-human antibody that has been modified to increase sequence homology with a human antibody, such that the antigen-binding properties of the antibody are retained but its in vivo Antigenicity decreased. Thus, the single chain trimeric CD40L Fc fusion proteins provided herein can comprise Fc monomers from humanized antibodies.

As used herein, the term "CD40 ligand" or "CD40L" refers to a protein or protein fragment that acts as a ligand for: CD40/TNFRSF5, the tumor necrosis factor receptor , costimulatory member of the TNFR) superfamily. Specific CD40L sequences are available from public access repositories, such as UniProt, eg UniProt accession number P29965. CD40L consists of an extracellular domain, a stalk region, a transmembrane helix, and a short cytoplasmic domain. The activity of CD40L is located in the 215 amino acid ectodomain, which is characterized by a sandwich structure consisting of a β-pleated plate, an α-helical loop, and a β-pleated plate. This structure allows trimerization of CD40L. The term CD40L further encompasses a polypeptide or any fragment thereof that has at least about 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 98, or at least 99% sequence identity and the ability to act as a ligand for CD40. The term CD40L encompasses full length CD40L and any fragment thereof, such as the extracellular domain portion of CD40L. The term further encompasses soluble CD40L (sCD40L), which has been reported in various disease settings and can exist in monomeric as well as multimeric forms. In the context of single-chain trimeric CD40L fusion proteins or single-chain trimeric CD40L Fc fusion proteins according to the present disclosure, individual CD40L components are referred to as "CD40L subunits". A portion of a fusion protein consisting of three CD40L subunits fused together may also be referred to as a "CD40L trimer".

As used herein, the term "fusion protein" refers to a protein or polypeptide that encompasses two or more peptide segments linked together to produce a protein that is not present in the same naturally occurring polypeptide. the sequence of.

As used herein, the term "single chain trimeric CD40L Fc fusion protein (single chain trimeric CD40L Fc fusion protein)" refers to a protein comprising: a monomeric Fc region (monomeric Fc region, mFc), which is usually linked to ( Optionally via a peptide tether, as described herein) to the three CD40L subunits (as described herein and shown in Figure 1). Specifically, the single-chain trimeric CD40L Fc fusion protein consists of three CD40L extracellular domains (ECD), or fragments thereof, linked to each other with a flexible peptide linker to form a linear CD40L trimer, which The linear CD40L trimer is linked to the N- or C-terminus of the Fc monomer peptide with a peptide tether. Thus, in various embodiments, the single-chain trimeric CD40L Fc fusion protein can be a C-terminal or N-terminal Fc fusion of the CD40L trimer.

In some embodiments, the CD40L trimer is linked to the N-terminus of the Fc monomer peptide. In some embodiments, the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 1 to 12, or a fragment thereof.

In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:1. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:2. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:3. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:4. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:5. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:6. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:7. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:8. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:9. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:10. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:11. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:12. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:1. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:2. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:3. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:4. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:5. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:6. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:7. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:8. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:9. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:10. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:11. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:12.

In some embodiments, the CD40L trimer is linked to the C-terminus of the Fc monomer peptide. In some embodiments, the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 13 to 19, or a fragment thereof. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO: 16 or a fragment thereof.

In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:13. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:14. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:15. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:16. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:17. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:18. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:19.

In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:13. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:14. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:15. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:16. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:17. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:18. In another embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:19.

In some embodiments, the CD40 ligand subunit comprises a portion of the CD40L extracellular domain. In some embodiments, the CD40L subunit comprises any one of the sequences selected from SEQ ID NOS: 20-22, or a fragment thereof.

In one embodiment, the CD40L subunit comprises a fragment of SEQ ID NO:20. In another embodiment, the CD40L subunit comprises a fragment of SEQ ID NO: 21. In another embodiment, the CD40L subunit comprises a fragment of SEQ ID NO: 22.

In one embodiment, the CD40L subunit comprises SEQ ID NO: 20. In another embodiment, the CD40L subunit comprises SEQ ID NO: 21. In another embodiment, the CD40L subunit comprises SEQ ID NO: 22.

In some cases, two single chain trimeric CD40L Fc fusion proteins can form a homodimeric Fc fusion protein or a heterodimeric Fc fusion protein, with the latter being preferred. In one embodiment, two single chain trimeric CD40L Fc fusion proteins form a homodimeric Fc fusion protein. In one embodiment, two single chain trimeric CD40L Fc fusion proteins form a heterodimeric Fc fusion protein. In some cases, one monomer of the heterodimeric Fc fusion protein comprises only Fc monomeric peptides or fragments thereof, and the other monomer is a single chain trimeric CD40L Fc fusion protein. In some embodiments, a single-chain trimeric CD40L Fc fusion protein can comprise a variant Fc monomer with one or more amino acid substitutions compared to a reference or wild-type Fc monomer. In some cases, the heterodimeric Fc fusion protein is one monomeric Fc monomer peptide or a fragment thereof, and a protein domain other than CD40L (such as a receptor, ligand, or other binding partner). Thus, a single chain trimeric CD40L Fc fusion protein may be a component of a heterodimeric Fc fusion protein which is a bispecific molecule.

The term "bispecific molecule" refers to a molecule having two binding domains, each of which is capable of specifically binding a target protein, ligand, or fragment thereof. Thus, while bispecific molecules can include binding domains (which are equivalent to non-antibody proteins), ligands and fragments thereof (including recombinant antigens), which can specifically bind another protein. In one embodiment, a bispecific molecule may comprise a single-chain trimeric CD40L Fc fusion protein.

The terms "binds/binding" refer to interactions between molecules, which include, for example, the formation of complexes. The term "binding domain" refers to the portion of a molecule that is responsible for the specific binding interaction with another molecule or ligand. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and/or van der Waals interactions. A complex may also include the association of two or more molecules held together by covalent or non-covalent bonds, interactions or forces. The strength of the total non-covalent interaction between a single antigen binding site on an antibody and a single epitope of a molecule of interest, such as an antigen, is the affinity of the antibody or functional fragment for that epitope. The ratio of the _off- rate (koff) to the on-rate ( _kon ) of a binding molecule (eg, an antibody) to a monovalent antigen (koff _{/ kon} ) is the dissociation constant, _KD , which is inversely related to affinity. The lower the _KD value, the higher the affinity of the binding molecule. Different complexes of binding molecules and their ligands (ie antibodies and antigens) have different _KD values depending on k _on and k _off . Binding domains can be 1×10 ⁻⁷ M or smaller (such as 1×10 ⁻⁸ M or smaller, 5×10 ⁻⁹ M or smaller, 1×10 ⁻⁹ M or smaller, 5×10 ⁻¹⁰ M or smaller, or 1×10 ⁻¹⁰ M or smaller) with a KD that specifically binds the target. The dissociation constant _KD of the binding molecules provided herein can be determined using any of the methods provided herein or any other method known to those of ordinary skill in the art. The affinity at a binding site does not always reflect the true strength of the interaction between a binding molecule and its binding partner.

Terms related to the binding molecules described herein, such as "bind to", "that specifically bind to", and the like are also used interchangeably herein and refer to specifically binding A binding molecule, such as a polypeptide, to the antigen binding domain of an antigen. A binding molecule or antigen binding domain that binds or specifically binds to an antigen may cross-react with the relevant antigen. In certain embodiments, a binding molecule or antigen binding domain that binds or specifically binds to an antigen does not cross-react with other antigens. Binding molecules or antigen-binding domains that bind or specifically bind to an antigen can be identified, for example, by immunoassays, ^Octet® , ^Biacore® , or other techniques known to those of ordinary skill in the art. In some embodiments, a binding molecule or antigen binding domain binds or specifically binds to an antigen when it binds the antigen with a higher affinity than any cross-reactive antigen, such as using experimental techniques such as radioimmunoassay assay (RIA) and enzyme-linked immunosorbent assay (ELISA)). Typically, a specific or selective reaction will be at least twice background signal or noise, and may be more than 10 times background. See, eg, Fundamental Immunology 332-36 (Paul ed., 2d ed. 1989) for a discussion of binding specificity. In certain embodiments, the extent of binding of a binding molecule or antigen binding domain to a "non-target" protein is less than about 10% of the binding of the binding molecule or antigen binding domain to its specific target antigen, e.g., as in fluorescence activated cell sorting (FACS) analysis or determined by RIA. Reference to terms such as "specific binding", "specifically binds to", or "is specific for" means that there is a measurable interaction with non-specific A combination of differences. For example, specific binding can be measured by determining the binding of a molecule compared to the binding of a control molecule (typically a molecule of similar structure that does not have binding activity). For example, specific binding can be determined by competition with a control molecule similar to the target (eg, excess non-labeled target). In this case, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by excess unlabeled target. A binding molecule or antigen binding domain that binds to an antigen includes one that is capable of binding an antigen with sufficient affinity such that the binding molecule is useful, eg, as a diagnostic agent targeting that antigen. In certain embodiments, the binding molecule or antigen binding domain that binds to the antigen has an equilibrium dissociation constant ( _KD ) of less than or equal to 800 nM, 600 nM, 550 nM, 500 nM, 300 nM, 250 nM, 100 nM, 50 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM. In certain embodiments, the binding molecule or antigen binding domain binds to a conserved epitope among antigens from different species (between human and cynomolgus monkey species).

"Binding affinity" generally refers to the sum total strength of non-covalent interactions between a single binding site of a molecule (eg, a binding protein, such as CD40L) and its binding partner (eg, CD40/TNFRSF5). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a binding molecule X for its binding partner Y can generally be expressed by the equilibrium dissociation constant ( _KD ). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen more rapidly and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which may be used for the purposes of the present disclosure. Specific illustrative examples include the following examples. In one embodiment, " _KD " or " _KD value" can be measured by assays known in the art (eg, by binding assays). _KD can be measured in RIA, eg, with a Fab version of the antibody of interest and its antigen (Chen et al. , 1999, J. Mol Biol 293:865-81). _KD or _KD values can also be measured by using biolayer interferometry (biolayer interferometry, BLI) or surface plasmon resonance (surface plasmon resonance, SPR) assay, which is obtained by Octet® (using e.g. Octet® Red96 system), or by Biacore® (using eg Biacore®TM-2000 or Biacore®TM-3000). "On-rate" or "rate of association" or "association rate" or "kon" can also be determined by the following: the same biolayer interferometry as above interferometry, BLI) or surface plasmon resonance (surface plasmon resonance, SPR) techniques using, for example, Octet® Red96, Biacore®TM-2000, or Biacore®TM-3000 systems.

In certain embodiments, the binding molecule or antigen-binding domain may comprise: portions of "humanized" forms of non-human (e.g., camelid, murine, non-human primate) antibodies, including those derived from human immune Sequences of globulins (e.g. recipient antibodies) in which the natural CDR residues may be replaced by residues from the corresponding CDRs from a non-human species (e.g. donor antibody) such as camelid, mouse, rat Mouse, rabbit, or non-human primate with desired specificity, affinity, and capacity. In some instances, one or more FR region residues of the human immunoglobulin sequence are replaced by corresponding non-human residues. In addition, humanized antibodies may comprise residues which are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine antibody potency. A humanized antibody heavy or light chain may comprise substantially all of at least one or more variable regions, wherein all or substantially all of the CDRs correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are These are human immunoglobulin sequences. In certain embodiments, a humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al. , 1986, Nature 321:522-25; Riechmann et al. , 1988, Nature 332:323-29; Presta, 1992, Curr. Op. Struct. Biol. 2:593-96 ; Carter et al. , 1992, Proc. Natl. Acad. Sci. USA 89:4285-89; US Patent Nos. 6,800,738; 6,719,971; 6,639,055; 6,407,213;

The techniques and procedures described or referred to herein include those that are generally well understood and/or commonly employed, using methods that are well known to those of ordinary skill in the art, such as, for example, the widely employed methods described by Sambrook et al. , Molecular Cloning: A Laboratory Manual (3d ed. 2001); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); and Antibody Engineering Vols 1 and 2 (Kontermann and Dübel eds., 2d ed. 2010).

Unless otherwise defined herein, the meanings of technical and scientific terms used in the embodiments of this application are those commonly understood by those skilled in the art. For the purpose of explaining this specification, the following descriptions of terms will be applied, and whenever appropriate, terms used in the singular will also include the plural and vice versa. In any event that any description of a presented term conflicts with any document incorporated herein by reference, the description of a presented term below will control.

In one aspect, provided herein is a polypeptide comprising a single-chain trimeric CD40L fusion protein. In some embodiments, the single-chain trimeric CD40L fusion protein comprises three CD40L subunits covalently linked to each other by a flexible peptide linker. In some embodiments, the single-chain trimeric CD40L fusion protein comprises three CD40L ECD domains, or fragments thereof. In specific embodiments, each CD40L subunit of the single-chain trimeric CD40L fusion protein comprises any one sequence selected from the group consisting of: CD40L v.1 (SEQ ID NO: 20) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL CD40L v.2 (SEQ ID NO: 21) QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL CD40L v.3 (SEQ ID NO: 22) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL CD40L v.4 (SEQ ID NO: 33) IAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL CD40L v.5 (SEQ ID NO: 34) QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

In one embodiment, the CD40L subunit of the single-chain trimeric CD40L fusion protein comprises SEQ ID NO: 20 or a fragment thereof. In another embodiment, the CD40L subunit of the single-chain trimeric CD40L fusion protein comprises SEQ ID NO: 21 or a fragment thereof. In another embodiment, the CD40L subunit of the single-chain trimeric CD40L fusion protein comprises SEQ ID NO: 22 or a fragment thereof. In another embodiment, the CD40L subunit of the single-chain trimeric CD40L fusion protein comprises SEQ ID NO: 33 or a fragment thereof. In another embodiment, the CD40L subunit of the single-chain trimeric CD40L fusion protein comprises SEQ ID NO: 34 or a fragment thereof. In one embodiment, the CD40L subunit of the single-chain trimeric CD40L fusion protein comprises SEQ ID NO:20. In another embodiment, the CD40L subunit of the single-chain trimeric CD40L fusion protein comprises SEQ ID NO: 21 or a fragment thereof. In another embodiment, the CD40L subunit of the single-chain trimeric CD40L fusion protein comprises SEQ ID NO: 22 or a fragment thereof. In another embodiment, the CD40L subunit of the single-chain trimeric CD40L fusion protein comprises SEQ ID NO: 33 or a fragment thereof. In another embodiment, the CD40L subunit of the single-chain trimeric CD40L fusion protein comprises SEQ ID NO: 34 or a fragment thereof. In one embodiment, the CD40L subunit of the single-chain trimeric CD40L fusion protein comprises SEQ ID NO:20. In another embodiment, the CD40L subunit of the single-chain trimeric CD40L fusion protein comprises SEQ ID NO: 21 or a fragment thereof. In another embodiment, the CD40L subunit of the single-chain trimeric CD40L fusion protein comprises SEQ ID NO: 22 or a fragment thereof. In another embodiment, the CD40L subunit of the single-chain trimeric CD40L fusion protein comprises SEQ ID NO: 33 or a fragment thereof. In another embodiment, the CD40L subunit of the single-chain trimeric CD40L fusion protein comprises SEQ ID NO: 34 or a fragment thereof.

In one embodiment, the single-chain trimeric CD40L fusion protein comprises a CD40L subunit having the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises a CD40L subunit having the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises a CD40L subunit having the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises a CD40L subunit having the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises a CD40L subunit having the amino acid sequence of SEQ ID NO:34.

In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; and a second CD40L subunit having the amine of SEQ ID NO:21 amino acid sequence. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; and a second CD40L subunit having the amine of SEQ ID NO:22 amino acid sequence. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; and a second CD40L subunit having the amine of SEQ ID NO:33 amino acid sequence. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; and a second CD40L subunit having the amine of SEQ ID NO:34 amino acid sequence. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; and a second CD40L subunit having the amine of SEQ ID NO:22 amino acid sequence. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; and a second CD40L subunit having the amine of SEQ ID NO:33 amino acid sequence. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; and a second CD40L subunit having the amine of SEQ ID NO:34 amino acid sequence. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; and a second CD40L subunit having the amine of SEQ ID NO:33 amino acid sequence. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; and a second CD40L subunit having the amine of SEQ ID NO:34 amino acid sequence. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; and a second CD40L subunit having the amine of SEQ ID NO:34 amino acid sequence.

In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having an amino acid sequence of SEQ ID NO:20; a second CD40L subunit having an amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20.

In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20.

In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having an amino acid sequence of SEQ ID NO:34; a second CD40L subunit having an amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:20.

In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having an amino acid sequence of SEQ ID NO:20; a second CD40L subunit having an amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having an amino acid sequence of SEQ ID NO:34; a second CD40L subunit having an amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:21.

In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having an amino acid sequence of SEQ ID NO:20; a second CD40L subunit having an amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having an amino acid sequence of SEQ ID NO:34; a second CD40L subunit having an amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:22.

In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having an amino acid sequence of SEQ ID NO:20; a second CD40L subunit having an amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having an amino acid sequence of SEQ ID NO:34; a second CD40L subunit having an amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:33.

In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:20; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having an amino acid sequence of SEQ ID NO:20; a second CD40L subunit having an amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:21; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:22; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:33; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having an amino acid sequence of SEQ ID NO:34; a second CD40L subunit having an amine group of SEQ ID NO:20 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:21 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:22 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:33 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34. In one embodiment, the single-chain trimeric CD40L fusion protein comprises: a first CD40L subunit having the amino acid sequence of SEQ ID NO:34; a second CD40L subunit having the amine group of SEQ ID NO:34 acid sequence; and the third CD40L subunit, which has the amino acid sequence of SEQ ID NO:34.

In some embodiments, the single-chain trimeric CD40L fusion protein comprises three CD40L subunits linked to each other with a flexible peptide linker to form a linear CD40L trimer. In a specific embodiment, the flexible peptide linker connecting the CD40L subunits in the fusion protein comprises a group selected from the group consisting of EGKSSGSGS (SEQ ID NO: 23) and (G ₃ S) _n (SEQ ID NO: 39) Any sequence, wherein n is an integer from 1 to 20. In some embodiments, the integer is one. In some embodiments, the integer is two. In some embodiments, the integer is three. In some embodiments, the integer is four. In some embodiments, the integer is 5. In some embodiments, the integer is six. In some embodiments, the integer is seven. In some embodiments, the integer is 8. In some embodiments, the integer is 9. In some embodiments, the integer is ten. In some embodiments, the integer is 11. In some embodiments, the integer is twelve. In some embodiments, the integer is 13. In some embodiments, the integer is fourteen. In some embodiments, the integer is 15. In some embodiments, the integer is 16. In some embodiments, the integer is 17. In some embodiments, the integer is 18. In some embodiments, the integer is 19. In some embodiments, the integer is 20.

In some embodiments, the flexible linker comprises EGKSSGSGS (SEQ ID NO: 23). In some embodiments, the flexible linker comprises (G ₃ S) ₃ (SEQ ID NO: 25). In some embodiments, the peptide linker comprises SEQ ID NO:23. In some embodiments, the peptide linker comprises SEQ ID NO:25.

In some embodiments, the peptide linker comprises the sequence EGKSSGSGS (SEQ ID NO: 23). In some embodiments, the peptide linker comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% of EGKSSGSGS (SEQ ID NO: 23) %, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity of amino acid sequences. In some embodiments, the peptide linker comprises amino acid sequences that are at least 50% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 55% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 60% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 65% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 70% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 75% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 80% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 85% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 90% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 95% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 98% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 99% identical. In some embodiments, the peptide linker comprises an amino acid sequence with 100% sequence identity.

In some embodiments _, the peptide linker comprises the sequence (G ₃ S) _n (SEQ ID NO: 39), _or comprises at least 50%, at least 55%, Amino acid sequences having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some embodiments, the peptide linker comprises amino acid sequences that are at least 50% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 55% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 60% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 65% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 70% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 75% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 80% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 85% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 90% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 95% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 98% identical. In some embodiments, the peptide linker comprises amino acid sequences that are at least 99% identical. In some embodiments, the peptide linker comprises an amino acid sequence with 100% sequence identity.

In a specific embodiment, the single-chain trimeric CD40L fusion protein comprises any sequence selected from the sequences shown in the following table, wherein the underlined part corresponds to the linker sequence: CD40L trimer v.1 (SEQ ID NO:35) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL CD40L trimer v.2 (SEQ ID NO:36) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL CD40L trimer v.3 (SEQ ID NO:37) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL CD40L trimer v.4 (SEQ ID NO:38) IAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGSQ IAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGSQ IAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

In one embodiment, the single chain trimeric CD40L fusion protein comprises SEQ ID NO:35. In another embodiment, the single chain trimeric CD40L fusion protein comprises SEQ ID NO:36. In another embodiment, the single chain trimeric CD40L fusion protein comprises SEQ ID NO:37. In another embodiment, the single chain trimeric CD40L fusion protein comprises SEQ ID NO:38.

In some embodiments, the single-chain trimeric CD40L fusion protein can also be recombinantly fused to, for example, a heterologous peptide or polypeptide (or a fragment thereof, e.g., to about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 amino acid polypeptides) to generate larger fusion proteins. Accordingly, in some embodiments, provided herein are polypeptides comprising: a CD40L trimer fused to a heterologous protein that is not derived from CD40L (which is sometimes referred to in this application as a "non-CD40L" peptide or polypeptide) peptide or polypeptide. The linkage between the CD40L trimer and the non-CD40L peptide or polypeptide can be directly or via a flexible peptide tether. In some embodiments, the non-CD40L peptide or polypeptide is fused to the N-terminus of the CD40L trimer. In some embodiments, the non-CD40L peptide or polypeptide is fused to the C-terminus of the CD40L trimer.

Accordingly, in one aspect, provided herein are fusion proteins comprising: a CD40L trimer fused to a non-CD40L peptide or polypeptide, either directly or via a flexible peptide tether. In some embodiments, the non-CD40L portion of the fusion protein comprises an Fc monomeric peptide.

Provided herein is a single chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and (b) an Fc monomeric peptide.

As provided herein, the single chain trimeric CD40L Fc fusion protein can comprise an Fc region from an IgG antibody. In some embodiments, the single chain trimeric CD40L Fc fusion protein can comprise an Fc region from an IgGl, IgG2, IgG3, or IgG4 antibody. In some embodiments, the single chain trimeric CD40L Fc fusion protein may comprise an Fc region from an IgGl antibody. In some embodiments, the single chain trimeric CD40L Fc fusion protein may comprise an Fc region from an IgG2 antibody. In some embodiments, the single chain trimeric CD40L Fc fusion protein may comprise an Fc region from an IgG3 antibody. In some embodiments, the single chain trimeric CD40L Fc fusion protein may comprise an Fc region from an IgG4 antibody. In some embodiments, the single chain trimeric CD40L Fc fusion protein may comprise: an Fc monomer that is a silent Fc domain or a modified Fc domain (with a genetically engineered Fc domain whose key mutations nullify Fc receptor binding and Eliminate antibody directed cellular cytotoxicity (ADCC) effector function).

In some embodiments, the trimeric CD40L portion of the fusion protein is genetically fused or chemically ligated to the C-terminus of the Fc region. In some embodiments, the trimeric CD40L portion of the fusion protein is genetically fused or chemically ligated to the N-terminus of the Fc region.

In some embodiments, the Fc monomer peptide is covalently linked to the CD40L trimer via a peptide tether.

As used herein, the term "tether" or "peptide tether" refers to the amino acid sequence linking the trimeric CD40L portion of the fusion protein to the Fc monomeric peptide. In some embodiments, the peptide tether comprises between 0 and 20 amino acids (inclusive). In some embodiments, the peptide tether comprises between 0 and 1 amino acids. In some embodiments, the peptide tether comprises between 0 and 5 amino acids. In some embodiments, the peptide tether comprises between 0 and 10 amino acids. In some embodiments, the peptide tether comprises between 0 and 15 amino acids. In some embodiments, the peptide tether comprises between 5 and 10 amino acids. In some embodiments, the peptide tether comprises between 5 and 15 amino acids. In some embodiments, the peptide tether comprises between 5 and 20 amino acids. In some embodiments, the peptide tether comprises between 10 and 15 amino acids. In some embodiments, the peptide tether comprises between 10 and 20 amino acids. In contrast, the term "linker" or "peptide linker" refers to the amino acid sequence that links CD40L subunits to each other to form a trimer.

In one embodiment, the peptide tether linking the trimeric CD40L portion of the fusion protein to the Fc monomeric peptide has the sequence (G ₄ S) _n (SEQ ID NO:40), where n is an integer from 1 to 20. In some embodiments, the integer is one. In some embodiments, the integer is two. In some embodiments, the integer is three. In some embodiments, the integer is four. In some embodiments, the integer is 5. In some embodiments, the integer is six. In some embodiments, the integer is seven. In some embodiments, the integer is 8. In some embodiments, the integer is 9. In some embodiments, the integer is ten. In some embodiments, the integer is 11. In some embodiments, the integer is twelve. In some embodiments, the integer is 13. In some embodiments, the integer is fourteen. In some embodiments, the integer is 15. In some embodiments, the integer is sixteen. In some embodiments, the integer is 17. In some embodiments, the integer is 18. In some embodiments, the integer is 19. In some embodiments, the integer is 20.

In one embodiment, the flexible tether does not comprise the (G ₄ S) (SEQ ID NO: 28) motif. In one embodiment, the flexible tether comprises (G ₄ S) ₁ (SEQ ID NO: 28). In one embodiment, the flexible tether comprises (G ₄ S) ₂ (SEQ ID NO: 26). In one embodiment, the flexible tether comprises (G ₄ S) ₃ (SEQ ID NO: 24). In one embodiment, the flexible tether comprises (G ₄ S) ₄ (SEQ ID NO: 27). In some embodiments, the peptide tether comprises SEQ ID NO:24. In some embodiments, the peptide tether comprises SEQ ID NO:26. In some embodiments, the peptide tether comprises SEQ ID NO:27. In some embodiments, the peptide tether comprises SEQ ID NO:28.

In some _embodiments , the peptide tether comprises the sequence (G ₄ S) _n (SEQ ID NO: 40), or comprises at least 50%, at least 55% _, Amino acid sequences having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some embodiments, the peptide linker comprises an amino acid sequence at least 50% identical to ( _G4S ) _n (SEQ ID NO:40). In some embodiments, the peptide linker comprises an amino acid sequence at least 55% identical to ( _G4S ) _n (SEQ ID NO: 40). In some embodiments, the peptide linker comprises an amino acid sequence at least 60% identical to ( _G4S ) _n (SEQ ID NO:40). In some embodiments, the peptide linker comprises an amino acid sequence at least 65% identical to ( _G4S ) _n (SEQ ID NO:40). In some embodiments, the peptide linker comprises an amino acid sequence at least 70% identical to ( _G4S ) _n (SEQ ID NO:40). In some embodiments, the peptide linker comprises an amino acid sequence at least 75% identical to ( _G4S ) _n (SEQ ID NO:40). In some embodiments, the peptide linker comprises an amino acid sequence at least 80% identical to ( _G4S ) _n (SEQ ID NO:40). In some embodiments, the peptide linker comprises an amino acid sequence at least 85% identical to ( _G4S ) _n (SEQ ID NO:40). In some embodiments, the peptide linker comprises an amino acid sequence at least 90% identical to ( _G4S ) _n (SEQ ID NO:40). In some embodiments, the peptide linker comprises an amino acid sequence at least 95% identical to ( _G4S ) _n (SEQ ID NO:40). In some embodiments, the peptide linker comprises an amino acid sequence at least 98% identical to ( _G4S ) _n (SEQ ID NO:40). In some embodiments, the peptide linker comprises an amino acid sequence at least 99% identical to ( _G4S ) _n (SEQ ID NO:40). In some embodiments, the peptide linker comprises an amino acid sequence having 100% sequence identity to (G ₄ S) _n (SEQ ID NO: 40).

In one embodiment, the peptide linker linking the CD40L subunits to each other to form the trimeric CD40L portion of the fusion protein may be a flexible linker comprising a flexible linker selected from the group consisting of EGKSSGSGS (SEQ ID NO: 23) and A sequence of the group consisting of (G ₃ S) _n (SEQ ID NO: 39), wherein n is an integer from 1 to 20. In some embodiments, the integer is one. In some embodiments, the integer is two. In some embodiments, the integer is three. In some embodiments, the integer is four. In some embodiments, the integer is 5. In some embodiments, the integer is six. In some embodiments, the integer is seven. In some embodiments, the integer is 8. In some embodiments, the integer is 9. In some embodiments, the integer is ten. In some embodiments, the integer is 11. In some embodiments, the integer is twelve. In some embodiments, the integer is 13. In some embodiments, the integer is fourteen. In some embodiments, the integer is 15. In some embodiments, the integer is sixteen. In some embodiments, the integer is 17. In some embodiments, the integer is 18. In some embodiments, the integer is 19. In some embodiments, the integer is 20. In one embodiment, the flexible linker comprises EGKSSGSGS (SEQ ID NO: 23). In one embodiment, the flexible linker comprises (G ₃ S) ₃ (SEQ ID NO: 25). In some embodiments, the peptide linker comprises SEQ ID NO:23. In some embodiments, the peptide linker comprises SEQ ID NO:25.

In some embodiments, the peptide tether comprises the sequence EGKSSGSGS (SEQ ID NO:23), or comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70% of the sequence EGKSSGSGS (SEQ ID NO:23) %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity of amino acid sequences. In some embodiments, the peptide linker comprises an amino acid sequence at least 50% identical to EGKSSGSGS (SEQ ID NO: 23). In some embodiments, the peptide linker comprises an amino acid sequence at least 55% identical to EGKSSGSGS (SEQ ID NO: 23). In some embodiments, the peptide linker comprises an amino acid sequence at least 60% identical to EGKSSGSGS (SEQ ID NO: 23). In some embodiments, the peptide linker comprises an amino acid sequence at least 65% identical to EGKSSGSGS (SEQ ID NO: 23). In some embodiments, the peptide linker comprises an amino acid sequence at least 70% identical to EGKSSGSGS (SEQ ID NO: 23). In some embodiments, the peptide linker comprises an amino acid sequence at least 75% identical to EGKSSGSGS (SEQ ID NO: 23). In some embodiments, the peptide linker comprises an amino acid sequence at least 80% identical to EGKSSGSGS (SEQ ID NO: 23). In some embodiments, the peptide linker comprises an amino acid sequence at least 85% identical to EGKSSGSGS (SEQ ID NO: 23). In some embodiments, the peptide linker comprises an amino acid sequence at least 90% identical to EGKSSGSGS (SEQ ID NO: 23). In some embodiments, the peptide linker comprises an amino acid sequence at least 95% identical to EGKSSGSGS (SEQ ID NO: 23). In some embodiments, the peptide linker comprises an amino acid sequence at least 98% identical to EGKSSGSGS (SEQ ID NO: 23). In some embodiments, the peptide linker comprises an amino acid sequence at least 99% identical to EGKSSGSGS (SEQ ID NO: 23). In some embodiments, the peptide linker comprises an amino acid sequence with 100% sequence identity to EGKSSGSGS (SEQ ID NO: 23).

In some embodiments _, the peptide linker comprises the sequence (G ₃ S) _n (SEQ ID NO: 39), _or comprises at least 50%, at least 55%, Amino acid sequences having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some embodiments, the peptide linker comprises an amino acid sequence at least 50% identical to (G ₃ S) _n (SEQ ID NO: 39). In some embodiments, the peptide linker comprises an amino acid sequence at least 55% identical to (G ₃ S) _n (SEQ ID NO: 39). In some embodiments, the peptide linker comprises an amino acid sequence at least 60% identical to (G ₃ S) _n (SEQ ID NO: 39). In some embodiments, the peptide linker comprises an amino acid sequence at least 65% identical to (G ₃ S) _n (SEQ ID NO: 39). In some embodiments, the peptide linker comprises an amino acid sequence at least 70% identical to (G ₃ S) _n (SEQ ID NO: 39). In some embodiments, the peptide linker comprises an amino acid sequence at least 75% identical to ( _G3S ) _n (SEQ ID NO: 39). In some embodiments, the peptide linker comprises an amino acid sequence at least 80% identical to (G ₃ S) _n (SEQ ID NO: 39). In some embodiments, the peptide linker comprises an amino acid sequence at least 85% identical to (G ₃ S) _n (SEQ ID NO: 39). In some embodiments, the peptide linker comprises an amino acid sequence at least 90% identical to (G ₃ S) _n (SEQ ID NO: 39). In some embodiments, the peptide linker comprises an amino acid sequence at least 95% identical to (G ₃ S) _n (SEQ ID NO: 39). In some embodiments, the peptide linker comprises an amino acid sequence at least 98% identical to (G ₃ S) _n (SEQ ID NO: 39). In some embodiments, the peptide linker comprises an amino acid sequence at least 99% identical to (G ₃ S) _n (SEQ ID NO: 39). In some embodiments, the peptide linker comprises an amino acid sequence having 100% sequence identity to (G ₃ S) _n (SEQ ID NO: 39).

In a specific embodiment, the Fc monomer in the fusion protein according to the present disclosure comprises any sequence selected from the following: Fc monomer v.1 (SEQ ID NO:29) VERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fc monomer v.2 (SEQ ID NO:30) EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fc monomer v.3 (SEQ ID NO:31) HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

In some embodiments, the CD40L trimer is linked to the N-terminus of the Fc monomer peptide. In some embodiments, the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 1 to 12, or a fragment thereof.

In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:1. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:2. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:3. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:4. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:5. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:6. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:7. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:8. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:9. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:10. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:11. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:12. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:1. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:2. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:3. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:4. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:5. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:6. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:7. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:8. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:9. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:10. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:11. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:12.

In some embodiments, the CD40L trimer is linked to the C-terminus of the Fc monomer peptide. In some embodiments, the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 13 to 19, or a fragment thereof. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO: 16 or a fragment thereof. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:13. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:14. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:15. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:16. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:17. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:18. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises SEQ ID NO:19. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:13. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:14. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:15. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:16. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:17. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:18. In one embodiment, the single chain trimeric CD40L Fc fusion protein comprises a fragment of SEQ ID NO:19.

In some embodiments, the CD40 ligand subunits comprise a portion of the CD40L extracellular domain. In some embodiments, the CD40L subunit comprises any one of the sequences selected from SEQ ID NOS: 20-22, or a fragment thereof. In one embodiment, the CD40L subunit comprises SEQ ID NO: 20. In one embodiment, the CD40L subunit comprises SEQ ID NO: 21. In one embodiment, the CD40L subunit comprises SEQ ID NO: 22. In one embodiment, the CD40L subunit comprises a fragment of SEQ ID NO:20. In one embodiment, the CD40L subunit comprises a fragment of SEQ ID NO: 21. In one embodiment, the CD40L subunit comprises a fragment of SEQ ID NO: 22.

In some cases, two single chain trimeric CD40L Fc fusion proteins can form a homodimeric Fc fusion protein or a heterodimeric Fc fusion protein, with the latter being preferred. In one embodiment, two single chain trimeric CD40L Fc fusion proteins form a homodimeric Fc fusion protein. In one embodiment, two single chain trimeric CD40L Fc fusion proteins form a heterodimeric Fc fusion protein. In some cases, one monomer of the heterodimeric Fc fusion protein comprises only Fc monomeric peptides or fragments thereof, and the other monomer is a single chain trimeric CD40L Fc fusion protein. In one embodiment, one monomer of the heterodimeric Fc fusion protein comprises only Fc monomer peptides, and the other monomer is a single chain trimeric CD40L Fc fusion protein. In one embodiment, one monomer of the heterodimeric Fc fusion protein comprises only fragments of Fc monomer peptides, and the other monomer is a single chain trimeric CD40L Fc fusion protein. In some embodiments, the single-chain trimeric CD40L Fc fusion protein comprises a variant Fc monomer with one or more amino acid substitutions compared to a reference or wild-type Fc monomer. In some embodiments, one monomer of the heterodimeric Fc fusion protein comprises an Fc monomer peptide or fragment thereof, and a protein domain other than CD40L (such as a receptor, ligand, or other binding partner). In one embodiment, one monomer of the heterodimeric Fc fusion protein comprises an Fc monomer peptide and a protein domain other than CD40L. In one embodiment, one monomer of the heterodimeric Fc fusion protein comprises a fragment of an Fc monomer peptide and a protein domain other than CD40L. In one embodiment, the protein domain other than CD40L is a receptor. In one embodiment, a protein domain other than CD40L is a ligand. In one embodiment, a protein domain other than CD40L is another binding partner. Thus, a single chain trimeric CD40L Fc fusion protein may be a component of a heterodimeric Fc fusion protein which is a bispecific molecule. Accordingly, in some embodiments, there is provided a bispecific molecule comprising a single-chain trimeric CD40L Fc fusion protein provided herein.

In some embodiments, the CD40L trimer system provided herein is recombinantly fused or chemically conjugated (covalently or non-covalently conjugated) to the antigen of interest. In one embodiment, the CD40L trimeric system provided herein is recombinantly fused to the target antigen. In one embodiment, a CD40L trimer provided herein is chemically conjugated to an antigen of interest. In one embodiment, chemical ligation is covalent ligation. In one embodiment, chemical ligation is non-covalent ligation. In some embodiments, the target antigen is recombinantly fused to the C-terminus of the CD40L trimer. In other embodiments, the target antigen is recombinantly fused to the N-terminus of the CD40L trimer. In some embodiments, the CD40L trimer and the antigen of interest are covalently linked via a peptide tether.

In specific embodiments, provided herein is a single chain trimeric CD40L target antigen fusion protein comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and (b) the target antigen.

In some embodiments, the antigen of interest is used to elicit an immune response. In some embodiments, the antigen of interest is derived from or derived from a pathogen or diseased cell. As used herein, a target antigen "derived" from a pathogen or diseased cell refers to a natural antigen produced by the pathogen or the diseased cell under naturally occurring or artificially produced conditions. Examples of antigens derived from pathogens or diseased cells include, but are not limited to, natural proteins expressed by pathogens or diseased cells in vivo or in vitro. As used herein, a target antigen "derived" from a pathogen or diseased cell refers to a variant of a natural antigen derived from the pathogen or diseased cell. For example, variants of proteins or peptides may result from one or more amino acid sequences (such as, for example, about 1 to about 25, about 1 to about 20, about 1 to about 15, about 1 to about 10, or about 1 to about 5). Variants of natural proteins may occur naturally (such as allelic or splice variants, or be fragmented or processed by immune cells), or may be artificially constructed. In some embodiments, the antigen of interest is derived from a pathogen. In some embodiments, the antigen of interest is derived from diseased cells. In some embodiments, the antigen of interest is derived from a pathogen. In some embodiments, the antigen of interest is derived from a diseased cell.

In some embodiments, the antigen of interest is derived from or derived from a protein expressed by an infecting pathogen, such as a virus, bacterium, fungus, or parasite. In one embodiment, the antigen of interest is derived from a protein expressed by an infectious pathogen. In one embodiment, the antigen of interest is derived from a protein expressed by an infecting pathogen. In one embodiment, the pathogenic system is a virus. In another embodiment, the pathogenic system is bacteria. In one embodiment, the pathogenic system is a fungus. In another embodiment, the pathogenic system is a parasite.

In some embodiments, the antigen of interest is derived from or derived from a protein expressed by a diseased host cell infected with an infecting pathogen. In one embodiment, the antigen of interest is derived from a protein expressed by diseased host cells. In one embodiment, the antigen of interest is derived from a protein expressed by a diseased host cell. In some embodiments, the diseased host cell line is infected with an infectious pathogen. In one embodiment, the pathogenic system is a virus. In another embodiment, the pathogenic system is bacteria. In one embodiment, the pathogenic system is a fungus. In another embodiment, the pathogenic system is a parasite.

In some embodiments, the antigen of interest is derived from or derived from a protein produced by an infecting pathogen. In some embodiments, the antigen of interest is derived from a protein produced by an infectious pathogen. In some embodiments, the antigen of interest is derived from a protein produced by an infecting pathogen. In some embodiments, infecting a pathogen mediates entry of the pathogen into the host. In one embodiment, the pathogenic system is a virus. In another embodiment, the pathogenic system is bacteria. In one embodiment, the pathogenic system is a fungus. In another embodiment, the pathogenic system is a parasite.

In some embodiments, the antigen of interest is derived from or derived from a protein produced by cancer cells or tumor stromal cells. In some embodiments, the antigen of interest is derived from a protein produced by cancer cells. In some embodiments, the antigen of interest is derived from a protein produced by tumor stromal cells. In some embodiments, the antigen of interest is derived from a protein produced by cancer cells. In some embodiments, the antigen of interest is derived from a protein produced by tumor stromal cells.

In a specific embodiment, the target antigen is a tumor associated antigen (tumor associated antigen, TAA) or an antigen fragment thereof. In one embodiment, the target antigen is TAA. In one embodiment, the target antigen is an antigenic fragment of TAA.

In some embodiments, the target antigen is recognized by the subject's innate immune system. In some embodiments, the antigen of interest is processed and presented by antigen presenting cells. In some embodiments, the antigen of interest is processed and presented in vitro by antigen presenting cells. In some embodiments, the antigen of interest is processed and presented in vivo by antigen presenting cells. In some embodiments, the antigen presenting cell line is B cells, dendritic cells, macrophages, natural killer cells, monocytes, granulocytes, eosinophils, neutrophils, or combinations thereof. In some embodiments, the antigen presenting cell line is dendritic cells. In some embodiments, the antigen presenting cell line is a B cell. In some embodiments, the antigen presenting cell line is dendritic cells. In some embodiments, the antigen presenting cell line is macrophages. In some embodiments, the antigen presenting cell line is a natural killer cell. In some embodiments, the antigen presenting cell line is monocytes. In some embodiments, the antigen presenting cell line pellets. In some embodiments, the antigen-presenting cell line eosinophils. In some embodiments, the antigen presenting cell line is a neutrophil.

In some embodiments, the antigen of interest is derived from a protein expressed on the surface of a cell, where the expression marks cells undergoing apoptosis (eg, via cell-mediated cytotoxicity or antibody-dependent cytotoxicity).

In some embodiments, the antigen of interest is derived from a protein expressed on the surface of a cell. In certain embodiments, a marker protein on the surface of a cell marks a cell undergoing apoptosis. In some embodiments, apoptosis is via cell-mediated cytotoxicity. In other embodiments, apoptosis is via antibody-dependent cellular cytotoxicity.

In some embodiments, the antigen of interest is derived from a protein expressed on the surface of a cell, wherein the expression marks a cell undergoing phagocytosis. In some embodiments, the target antigen is derived from a protein specifically recognized by circulating antibodies in the subject.

In some embodiments, the single-chain trimeric CD40L fusion protein can also be chemically conjugated (covalently or non-covalently bound) or recombinantly fused to, for example, a diagnostic or detectable agent. In one embodiment, the single-chain trimeric CD40L fusion protein is chemically joined to another molecule. In one embodiment, joining is covalent joining. In another embodiment, the ligation is non-covalent. In one embodiment, the single-chain trimeric CD40L fusion protein is recombinantly fused to another molecule. In some embodiments, the other molecule is a diagnostic agent. In some embodiments, another molecule is a detectable agent. In specific embodiments, the single-chain trimeric CD40L fusion protein of the present disclosure is coupled to a detectable substance, which includes but is not limited to various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphate Enzymes, β-galactosidase, or acetylcholinesterase; prosthetic groups such as but not limited to streptavidin/biotin or avidin/biotin; fluorescent materials such as but not limited to umbelliferone, Luciferin, fluorescent isothiocyanate, rose bengal, dichlorotrimethamine luciferin, dansyl chloride, or phycoerythrin; luminescent materials such as, but not limited to, luminol; bioluminescence Materials such as but not limited to luciferase, luciferin, or aequorin; chemiluminescent materials such as but not limited to acridine-based compounds or HALOTAG; radioactive materials such as but not limited to iodine ( ^131I , ¹²⁵ I, ¹²³ I, and ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹⁵ In, ¹¹³ In, ¹¹² In, and ¹¹¹ In), 鎝 ( ⁹⁹ Tc) , thallium ( ²⁰¹ Ti), gallium ( ⁶⁸ Ga and ⁶⁷ Ga), palladium ( ¹⁰³ Pd), molybdenum ( ⁹⁹ Mo), xenon ( ¹³³ Xe), fluorine ( ¹⁸ F), ¹⁵³ Sm, ¹⁷⁷ Lu, ¹⁵⁹ Gd, ¹⁴⁹ Pm, ¹⁴⁰ La, ¹⁷⁵ Yb, ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, 186 Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Ru, ⁶⁸ Ge, ⁵⁷ Co, ⁶⁵ Zn, ^{85 Sr} , ³² P, ¹⁵³ Gd , ¹⁶⁹ Yb, ⁵¹ Cr, ⁵⁴ Mn, ⁷⁵ Se, ¹¹³ Sn, or ¹¹⁷ Sn; positron-emitting metals, which use various positron emission tomography techniques; and nonradioactive paramagnetic metal ions.

In some embodiments, the fusion proteins provided herein can be fused to a marker or "tag" sequence, such as a peptide, to facilitate purification. In specific embodiments, the marker or tag amino acid sequence is a hexahistidine peptide, such as the tag provided in the pQE vector (see eg, QIAGEN, Inc.) and the like (many of which are commercially available). For example, as described in Gentz et al. , 1989, Proc. Natl. Acad. Sci. USA 86:821-24, hexahistidine can be used for purification of conventional fusion proteins. Other peptide tags useful for purification include, but are not limited to: hemagglutinin ("HA") tags, which correspond to epitopes derived from the influenza hemagglutinin protein (Wilson et al. , 1984, Cell 37:767- 78); and the "FLAG" label.

Methods for fusing or joining moieties, including polypeptides, are known (see, e.g., Arnon et al. , Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy , in Monoclonal Antibodies and Cancer Therapy 243-56 (Reisfeld et al. eds. , 1985); Hellstrom et al. , Antibodies for Drug Delivery , in Controlled Drug Delivery 623-53 (Robinson et al. eds., 2d ed. 1987); Thorpe, Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review , in Monoclonal Antibodies: Biological and Clinical Applications 475-506 (Pinchera et al. eds., 1985); Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy , in Monoclonal Antibodies for Cancer Detection and-1 Therapy 303 (Baldwin et al. eds., 1985); Thorpe et al. , 1982, Immunol. Rev. 62:119-58; US Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,723,125; 5,783,181; 5,908,626; 5,844,095; and 5,112,946; EP 307,434; EP 367,166; EP 394,827; /34631, and WO 99/04813; Ashkenazi et al. , 1991, Proc. Natl . Acad. Sci. USA, 88: 10535-39; Traunecker et al. , 1988, Nature, 331:84-86; Zheng et al. , 1995, J. Immunol. 154:5590-600; and Vil et al. , 1992, Proc. Natl. Acad. Sci. USA 89:11337-41).

In some embodiments, a polypeptide comprising a single-chain trimeric CD40L fusion protein provided herein modulates CD40 when bound to CD40. In certain embodiments, a polypeptide comprising a single-chain trimeric CD40L fusion protein provided herein activates CD40 when bound to CD40.

In one aspect, CD40 is activated upon binding to a single-chain trimeric CD40L fusion protein. In some embodiments, the single chain trimeric CD40L fusion protein activates CD40 with an _EC50 of less than about 4 nM. In some embodiments, the single-chain trimeric CD40L fusion protein activates CD40 with an _EC50 of less than about 3 nM. In some embodiments, the single-chain trimeric CD40L fusion protein activates CD40 with an _EC50 of less than about 1 nM. In some embodiments, the single chain trimeric CD40L fusion protein activates CD40 with an _EC50 of less than about 500 pM. In some embodiments, the single-chain trimeric CD40L fusion protein activates CD40 with an _EC50 of less than about 100 pM.

In one aspect, CD40 is activated upon binding to a single-chain trimeric CD40L Fc fusion protein. In some embodiments, the single chain trimeric CD40L Fc fusion protein activates CD40 with an _EC50 of less than about 4 nM. In some embodiments, the single chain trimeric CD40L Fc fusion protein activates CD40 with an _EC50 of less than about 3 nM. In some embodiments, the single chain trimeric CD40L Fc fusion protein activates CD40 with an _EC50 of less than about 1 nM. In some embodiments, the single chain trimeric CD40L Fc fusion protein activates CD40 with an _EC50 of less than about 500 pM. In some embodiments, the single chain trimeric CD40L Fc fusion protein activates CD40 with an _EC50 of less than about 100 pM.

In certain embodiments, the _EC50 is less than about 1 nM. In one embodiment, the _EC50 is less than about 0.9 nM. In one embodiment, the _EC50 is less than about 0.8 nM. In one embodiment, the _EC50 is less than about 0.7 nM. In one embodiment, the _EC50 is less than about 0.6 nM. In one embodiment, the _EC50 is less than about 0.5 nM. In one embodiment, the _EC50 is less than about 0.4 nM. In one embodiment, the _EC50 is less than about 0.300 nM. In one embodiment, the _EC50 is less than about 0.2 nM. In one embodiment, the _EC50 is less than about 0.19 nM. In one embodiment, the _EC50 is less than about 0.18 nM. In one embodiment, the _EC50 is less than about 0.17 nM. In one embodiment, the _EC50 is less than about 0.16 nM. In one embodiment, the _EC50 is less than about 0.15 nM. In one embodiment, the _EC50 is less than about 0.14 nM. In one embodiment, the _EC50 is less than about 0.13 nM. In one embodiment, the _EC50 is less than about 0.12 nM. In one embodiment, the _EC50 is less than about 0.11 nM. In one embodiment, the _EC50 is less than about 0.1 nM. In one embodiment, the _EC50 is less than about 0.09 nM. In one embodiment, the _EC50 is less than about 0.08 nM. In one embodiment, the _EC50 is less than about 0.07 nM. In one embodiment, the _EC50 is less than about 0.06 nM. In one embodiment, the _EC50 is less than about 0.05 nM. In one embodiment, the _EC50 is less than about 0.04 nM. In one embodiment, the _EC50 is less than about 0.03 nM. In one embodiment, the _EC50 is less than about 0.02 nM. In one embodiment, the _EC50 is at or less than about 0.01 nM. In some embodiments. In one embodiment, _{the EC50} is less than about 1 pM. In one embodiment, the _EC50 is less than about 0.9 pM. In one embodiment, the _EC50 is less than about 0.8 pM. In one embodiment, the _EC50 is less than about 0.7 pM. In one embodiment, the _EC50 is less than about 0.6 pM. In one embodiment, the _EC50 is less than about 0.5 pM. In one embodiment, the _EC50 is less than about 0.4 pM. In one embodiment, the _EC50 is less than about 0.300 pM. In one embodiment, the _EC50 is less than about 0.2 pM. In one embodiment, the _EC50 is less than about 0.19 pM. In one embodiment, the _EC50 is less than about 0.18 pM. In one embodiment, the _EC50 is less than about 0.17 pM. In one embodiment, the _EC50 is less than about 0.16 pM. In one embodiment, the _EC50 is less than about 0.15 pM. In one embodiment, the _EC50 is less than about 0.14 pM. In one embodiment, the _EC50 is less than about 0.13 pM. In one embodiment, the _EC50 is less than about 0.12 pM. In one embodiment, the _EC50 is less than about 0.11 pM. In one embodiment, the _EC50 is less than about 0.1 pM. In one embodiment, the _EC50 is less than about 0.09 pM. In one embodiment, the _EC50 is less than about 0.08 pM. In one embodiment, the _EC50 is less than about 0.07 pM. In one embodiment, the _EC50 is less than about 0.06 pM. In one embodiment, the _EC50 is less than about 0.05 pM. In one embodiment, the _EC50 is less than about 0.04 pM. In one embodiment, the _EC50 is less than about 0.03 pM. In one embodiment, the _EC50 is less than about 0.02 pM. In one embodiment, the _EC50 is at or less than about 0.01 pM. In some embodiments. In one embodiment, the _EC50 is less than about 1000 pM. In one embodiment, the _EC50 is less than about 900 pM. In one embodiment, the _EC50 is less than about 800 pM. In one embodiment, the _EC50 is less than about 700 pM. In one embodiment, the _EC50 is less than about 600 pM. In one embodiment, the _EC50 is less than about 500 pM. In one embodiment, the _EC50 is less than about 400 pM. In one embodiment, the _EC50 is less than about 300 pM. In one embodiment, the _EC50 is less than about 200 pM. In one embodiment, the _EC50 is less than about 190 pM. In one embodiment, the _EC50 is less than about 180 pM. In one embodiment, the _EC50 is less than about 170 pM. In one embodiment, the _EC50 is less than about 160 pM. In one embodiment, the _EC50 is less than about 150 pM. In one embodiment, the _EC50 is less than about 140 pM. In one embodiment, the _EC50 is less than about 130 pM. In one embodiment, the _EC50 is less than about 120 pM. In one embodiment, the _EC50 is less than about 110 pM. In one embodiment, the _EC50 is less than about 100 pM. In one embodiment, the _EC50 is less than about 90 pM. In one embodiment, the _EC50 is less than about 80 pM. In one embodiment, the _EC50 is less than about 70 pM. In one embodiment, the _EC50 is less than about 60 pM. In one embodiment, the _EC50 is less than about 50 pM. In one embodiment, the _EC50 is less than about 40 pM. In one embodiment, the _EC50 is less than about 30 pM. In one embodiment, the _EC50 is less than about 20 pM. In one embodiment, the _EC50 is less than about 10 pM.

Also provided herein are methods of modulating a CD40 polypeptide comprising contacting a CD40 polypeptide with a polypeptide comprising a single-chain trimeric CD40L fusion protein provided herein. Also provided herein are methods of activating a CD40 polypeptide comprising contacting a CD40 polypeptide with a polypeptide comprising a single-chain trimeric CD40L fusion protein provided herein.

Also provided is a method of activating a CD40 polypeptide comprising contacting the CD40 polypeptide with a single-chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits whose elements are covalently linked to each other by a peptide linker; and (b) an Fc monomeric peptide, wherein the single chain trimeric CD40L Fc fusion protein activates the CD40 polypeptide upon binding.

Also provided is a method of activating a target cell expressing a CD40 polypeptide comprising contacting the target cell with a polypeptide comprising a single-chain trimeric CD40L fusion protein according to the present disclosure, thereby producing an activated target cell. In a specific embodiment, the polypeptide in contact with the target cell is a single chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and (b) An Fc monomeric peptide, wherein the polypeptide activates target cells upon binding to CD40. In some embodiments, the activated target cell in turn activates another cell. In some embodiments, T cells are activated by activating target cells. In some embodiments, activated target cells activate a population of T cells.

In some embodiments, the cell line of interest is an antigen presenting cell. In some embodiments, the target cell is an immune cell. In some embodiments, the target cell line is B cells, natural killer cells, dendritic cells, macrophages, monocytes, granulocytes, eosinophils, neutrophils, or combinations thereof. In one embodiment, the target cell is a dendritic cell. In one embodiment, the cell of interest is a macrophage. In one embodiment, the target cell is a natural killer cell. In one embodiment, the cell of interest is a mononuclear spheroid. In one embodiment, the cell of interest is a pellet. In one embodiment, the target cell line is eosinophilic spheres. In one embodiment, the cell of interest is a pellet. In one embodiment, the cell of interest is a neutrophil. In some embodiments, the target cell is a Langerhans cell. In some embodiments, the cell of interest is a prostate cell. In some embodiments, the target cell is a B cell. In some embodiments, the target cell is a naive B cell. In some embodiments, the cell of interest is a memory B cell. In some embodiments, the target cell is a basal respiratory cell.

In some embodiments, activating a target cell activates another cell. In some embodiments, the activated target cell activates another cell that does not express a CD40 polypeptide. In some embodiments, immune cells are activated by activating target cells. In some embodiments, T cells are activated by activating target cells. In some embodiments, activated target cells activate CD4+ T cells, CD8+ T cells, MAIT, natural killer cells, neutrophils, or combinations thereof. In one embodiment, activated target cells activate CD4+ T cells. In one embodiment, activated target cells activate CD8+ T cells. In one embodiment, activated target cells activate MAIT. In one embodiment, the activated target cell is a natural killer cell. In one embodiment, activated target cells activate neutrophils.

In some embodiments, activation of the target cell is measured as increased proliferation or maturation of the target cell. In one embodiment, activation of the target cell is measured as increased proliferation of the target cell. In one embodiment, the activated line of the target cell is measured as increased maturation of the target cell. In specific embodiments, the proliferation or maturation of the target cell is increased by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%. In one embodiment, the proliferative lineage of the target cell is increased by about 10 to 100%. In another embodiment, the proliferative lineage of the target cell is increased by about 100 to 200%. In another embodiment, the proliferative lineage of the target cell is increased by about 200 to 300%. In another embodiment, the proliferative lineage of the target cell is increased by about 300 to 400%. In another embodiment, the proliferative lineage of the target cell is increased by about 400 to 500%. In another embodiment, the proliferative lineage of the target cell is increased by about 500 to 600%. In another embodiment, the proliferative lineage of the target cell is increased by about 600 to 700%. In another embodiment, the proliferative lineage of the target cell is increased by about 700 to 800%. In another embodiment, the proliferative lineage of the target cell is increased by about 800 to 900%. In another embodiment, the proliferative lineage of the target cell is increased by about 900 to 1000%. In one embodiment, the proliferative lineage of the target cell is increased by about 10%. In one embodiment, the proliferative lineage of the target cell is increased by about 20%. In one embodiment, the proliferative lineage of the target cell is increased by about 30%. In one embodiment, the proliferative lineage of the target cell is increased by about 40%. In one embodiment, the proliferative lineage of the target cell is increased by about 45%. In one embodiment, the proliferative lineage of the target cell is increased by about 50%. In one embodiment, the proliferative lineage of the target cell is increased by about 55%. In one embodiment, the proliferative lineage of the target cell is increased by about 60%. In one embodiment, the proliferative lineage of the target cell is increased by about 65%. In one embodiment, the proliferative lineage of the target cell is increased by about 70%. In one embodiment, the proliferative lineage of the target cell is increased by about 75%. In one embodiment, the proliferative lineage of the target cell is increased by about 80%. In one embodiment, the proliferative lineage of the target cell is increased by about 85%. In one embodiment, the proliferative lineage of the target cell is increased by about 90%. In one embodiment, the proliferative lineage of the target cell is increased by about 95%. In one embodiment, the proliferative lineage of the target cell is increased by about 100%. In one embodiment, the proliferative lineage of the target cell is increased by about 125%. In one embodiment, the proliferative lineage of the target cell is increased by about 150%. In one embodiment, the proliferative lineage of the target cell is increased by about 175%. In one embodiment, the proliferative lineage of the target cell is increased by about 200%. In one embodiment, the proliferative lineage of the target cell is increased by about 250%. In one embodiment, the proliferative lineage of the target cell is increased by about 300%. In one embodiment, the proliferative lineage of the target cell is increased by about 400%. In one embodiment, the proliferative lineage of the target cell is increased by about 500%. In one embodiment, the proliferative lineage of the target cell is increased by about 600%. In one embodiment, the proliferative lineage of the target cell is increased by about 700%. In one embodiment, the proliferative lineage of the target cell is increased by about 800%. In one embodiment, the proliferative lineage of the target cell is increased by about 900%. In one embodiment, the proliferative lineage of the target cell is increased by about 1000%. In one embodiment, the mature lineage of the cells of interest is increased by about 10 to 100%. In another embodiment, the mature lineage of the cells of interest is increased by about 100 to 200%. In another embodiment, the mature lineage of the cells of interest is increased by about 200 to 300%. In another embodiment, the mature lineage of the cells of interest is increased by about 300 to 400%. In another embodiment, the mature lineage of the cells of interest is increased by about 400 to 500%. In another embodiment, the mature lineage of the cells of interest is increased by about 500 to 600%. In another embodiment, the mature lineage of the cells of interest is increased by about 600 to 700%. In another embodiment, the mature lineage of the cells of interest is increased by about 700 to 800%. In another embodiment, the mature lineage of the cells of interest is increased by about 800 to 900%. In another embodiment, the mature lineage of the cells of interest is increased by about 900 to 1000%. In one embodiment, the mature lineage of the cells of interest is increased by about 10%. In one embodiment, the mature lineage of the cells of interest is increased by about 20%. In one embodiment, the mature lineage of the cells of interest is increased by about 30%. In one embodiment, the mature lineage of the cells of interest is increased by about 40%. In one embodiment, the mature lineage of the cells of interest is increased by about 45%. In one embodiment, the mature lineage of the cells of interest is increased by about 50%. In one embodiment, the mature lineage of the cells of interest is increased by about 55%. In one embodiment, the mature lineage of the cells of interest is increased by about 60%. In one embodiment, the mature lineage of the cells of interest is increased by about 65%. In one embodiment, the mature lineage of the cells of interest is increased by about 70%. In one embodiment, the mature lineage of the cells of interest is increased by about 75%. In one embodiment, the mature lineage of the cells of interest is increased by about 80%. In one embodiment, the mature lineage of the cells of interest is increased by about 85%. In one embodiment, the mature lineage of the cells of interest is increased by about 90%. In one embodiment, the mature lineage of the cells of interest is increased by about 95%. In one embodiment, the mature lineage of the cells of interest is increased by about 100%. In one embodiment, the mature lineage of the cells of interest is increased by about 125%. In one embodiment, the mature lineage of the cells of interest is increased by about 150%. In one embodiment, the mature lineage of the cells of interest is increased by about 175%. In one embodiment, the mature lineage of the cells of interest is increased by about 200%. In one embodiment, the mature lineage of the cells of interest is increased by about 250%. In one embodiment, the mature lineage of the cells of interest is increased by about 300%. In one embodiment, the mature lineage of the cells of interest is increased by about 400%. In one embodiment, the mature lineage of the cells of interest is increased by about 500%. In one embodiment, the mature lineage of the cells of interest is increased by about 600%. In one embodiment, the mature lineage of the cells of interest is increased by about 700%. In one embodiment, the mature lineage of the cells of interest is increased by about 800%. In one embodiment, the mature lineage of the cells of interest is increased by about 900%. In one embodiment, the mature lineage of the cells of interest is increased by about 1000%.

In some embodiments, activation of the target cell is measured as an extended survival time of the target cell. In specific embodiments, the survival time of the target cells is increased by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000 %. In one embodiment, the survival time of the target cells is increased by about 10 to 100%. In another embodiment, the survival time of the target cells is increased by about 100 to 200%. In another embodiment, the survival time of the target cells is increased by about 200 to 300%. In another embodiment, the survival time of the target cells is increased by about 300 to 400%. In another embodiment, the survival time of the target cells is increased by about 400 to 500%. In another embodiment, the survival time of the target cells is increased by about 500 to 600%. In another embodiment, the survival time of the target cells is increased by about 600 to 700%. In another embodiment, the survival time of the target cells is increased by about 700 to 800%. In another embodiment, the survival time of the target cells is increased by about 800 to 900%. In another embodiment, the survival time of the target cells is increased by about 900 to 1000%. In one embodiment, the survival time of the target cells is increased by about 10%. In one embodiment, the survival time of the target cells is increased by about 20%. In one embodiment, the survival time of the target cells is increased by about 30%. In one embodiment, the survival time of the target cells is increased by about 40%. In one embodiment, the survival time of the target cells is increased by about 45%. In one embodiment, the survival time of the target cells is increased by about 50%. In one embodiment, the survival time of the target cells is increased by about 55%. In one embodiment, the survival time of the target cells is increased by about 60%. In one embodiment, the survival time of the target cells is increased by about 65%. In one embodiment, the survival time of the target cells is increased by about 70%. In one embodiment, the survival time of the target cells is increased by about 75%. In one embodiment, the survival time of the target cells is increased by about 80%. In one embodiment, the survival time of the target cells is increased by about 85%. In one embodiment, the survival time of the target cells is increased by about 90%. In one embodiment, the survival time of the target cells is increased by about 95%. In one embodiment, the survival time of the target cells is increased by about 100%. In one embodiment, the survival time of the target cells is increased by about 125%. In one embodiment, the survival time of the target cells is increased by about 150%. In one embodiment, the survival time of the target cells is increased by about 175%. In one embodiment, the survival time of the target cells is increased by about 200%. In one embodiment, the survival time of the target cells is increased by about 250%. In one embodiment, the survival time of the target cells is increased by about 300%. In one embodiment, the survival time of the target cells is increased by about 400%. In one embodiment, the survival time of the target cells is increased by about 500%. In one embodiment, the survival time of the target cells is increased by about 600%. In one embodiment, the survival time of the target cells is increased by about 700%. In one embodiment, the survival time of the target cells is increased by about 800%. In one embodiment, the survival time of the target cells is increased by about 900%. In one embodiment, the survival time of the target cells is increased by about 1000%.

Also provided are methods of activating a T cell comprising contacting the T cell with an antigen presenting cell in the presence of a single-chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits , which are covalently linked to each other by a peptide linker; and (b) Fc monomeric peptides, wherein antigen presenting cells express a CD40 polypeptide, wherein the single chain trimeric CD40L Fc fusion protein activates T cells upon binding to the CD40 polypeptide. In some embodiments, antigen presenting cells present antigens to T cells.

In some embodiments, activation of a T cell population is measured by increased secretion of pro-inflammatory cytokines by T cells. In some embodiments, the pro-inflammatory cytokines are selected from the group consisting of IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, or any combination thereof. In one embodiment, the interleukin is IL-1. In one embodiment, the interleukin is IL-2. In one embodiment, the interleukin is IL-6. In one embodiment, the interleukin is IL-12. In one embodiment, the interleukin is IL-17. In one embodiment, the interleukin is IL-22. In one embodiment, the interleukin is IL-23. In one embodiment, the cytokine is GM-CSF. In one embodiment, the cytokine is TNF-α. In one embodiment, the interleukin is IFN-γ.

In some embodiments, antigen presenting cells present antigens to T cells. In some embodiments, upon activation of the antigen presenting cells by the single-chain trimeric CD40L Fc fusion protein, antigen presentation by the antigen presenting cells to T cells is increased. In some embodiments, the population of antigen-presenting cells comprises B cells, dendritic cells, macrophages, natural killer cells, monocytes, granulocytes, eosinophils, neutrophils, or combinations thereof. In some embodiments, the population of antigen presenting cells comprises B cells. In some embodiments, the population of antigen presenting cells comprises macrophages. In some embodiments, the population of antigen presenting cells comprises dendritic cells. In some embodiments, the population of antigen presenting cells comprises natural killer cells. In some embodiments, the population of antigen presenting cells comprises monocytes. In some embodiments, the population of antigen presenting cells comprises spheroids. In some embodiments, the population of antigen-presenting cells comprises eosinophils. In some embodiments, the population of antigen presenting cells comprises neutrophils.

Also provided are methods of activating dendritic cells comprising contacting a CD40 polypeptide with a single-chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits that are separated by peptide the linkers are covalently linked to each other; and (b) an Fc monomeric peptide, wherein the single chain trimeric CD40L Fc fusion protein activates the dendritic cell polypeptide upon binding CD40.

In some embodiments, the methods comprise contacting the CD40 polypeptide with a dimer comprising two single chain trimeric CD40L Fc fusion proteins each comprising: (a) three CD40L subunits, which are covalently linked to each other by a peptide linker; and (b) an Fc monomer peptide.

In some embodiments, provided herein is a method for promoting antibody production in a population of B cells comprising contacting the B cells with a polypeptide comprising a single-chain trimeric CD40L fusion protein according to the disclosure. In a specific embodiment, the polypeptide contacting the B cell population is a single chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and (b ) Fc monomer peptide.

In some embodiments, B cells are contacted with a polypeptide comprising a single-chain trimeric CD40L fusion protein in the presence of an antigen of interest, and wherein the antibodies produced by the B cells specifically bind to the antigen. In some embodiments, the antigen of interest is chemically conjugated or recombinantly fused to a single-chain trimeric CD40L fusion protein. In some embodiments, the antigen of interest is presented by an antigen presenting cell. In some embodiments, the antigen of interest is associated with an MHC class I complex. In some embodiments, the antigen of interest is associated with an MHC class II complex. In some embodiments, the antigen of interest is derived from or derived from an infectious pathogen. In some embodiments, the antigen of interest is derived from or derived from a diseased cell. In some embodiments, the diseased cell is a cancer cell. In some embodiments, the diseased cell line is a cell infected with an infectious pathogen. In some embodiments, the infection pathogenic system is a virus, bacterium, fungus, or parasite.

In some embodiments, antibody production by a B cell population is increased by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% , 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%. In one embodiment, antibody production by a B cell population is increased by about 10 to 100%. In another embodiment, antibody production by a B cell population is increased by about 100 to 200%. In another embodiment, antibody production by the B cell population is increased by about 200 to 300%. In another embodiment, antibody production by the B cell population is increased by about 300 to 400%. In another embodiment, antibody production by the B cell population is increased by about 400 to 500%. In another embodiment, antibody production by the B cell population is increased by about 500 to 600%. In another embodiment, antibody production by the B cell population is increased by about 600 to 700%. In another embodiment, antibody production by the B cell population is increased by about 700 to 800%. In another embodiment, antibody production by the B cell population is increased by about 800 to 900%. In another embodiment, antibody production by the B cell population is increased by about 900 to 1000%. In one embodiment, antibody production by the B cell population is increased by about 10%. In one embodiment, antibody production by the B cell population is increased by about 20%. In one embodiment, antibody production by the B cell population is increased by about 30%. In one embodiment, antibody production by the B cell population is increased by about 40%. In one embodiment, antibody production by the B cell population is increased by about 45%. In one embodiment, antibody production by the B cell population is increased by about 50%. In one embodiment, antibody production by the B cell population is increased by about 55%. In one embodiment, antibody production by the B cell population is increased by about 60%. In one embodiment, antibody production by the B cell population is increased by about 65%. In one embodiment, antibody production by the B cell population is increased by about 70%. In one embodiment, antibody production by the B cell population is increased by about 75%. In one embodiment, antibody production by the B cell population is increased by about 80%. In one embodiment, antibody production by the B cell population is increased by about 85%. In one embodiment, antibody production by the B cell population is increased by about 90%. In one embodiment, antibody production by the B cell population is increased by about 95%. In one embodiment, antibody production by a B cell population is increased by about 100%. In one embodiment, antibody production by the B cell population is increased by about 125%. In one embodiment, antibody production by the B cell population is increased by about 150%. In one embodiment, antibody production by the B cell population is increased by about 175%. In one embodiment, antibody production by the B cell population is increased by about 200%. In one embodiment, antibody production by the B cell population is increased by about 250%. In one embodiment, antibody production by the B cell population is increased by about 300%. In one embodiment, antibody production by the B cell population is increased by about 400%. In one embodiment, antibody production by the B cell population is increased by about 500%. In one embodiment, antibody production by the B cell population is increased by about 600%. In one embodiment, antibody production by the B cell population is increased by about 700%. In one embodiment, antibody production by the B cell population is increased by about 800%. In one embodiment, antibody production by the B cell population is increased by about 900%. In one embodiment, antibody production by the B cell population is increased by about 1000%. In one embodiment, antibody production by a B cell population is increased by about 10 to 100%. In another embodiment, antibody production by a B cell population is increased by about 100 to 200%. In another embodiment, antibody production by the B cell population is increased by about 200 to 300%. In another embodiment, antibody production by the B cell population is increased by about 300 to 400%. In another embodiment, antibody production by the B cell population is increased by about 400 to 500%. In another embodiment, antibody production by the B cell population is increased by about 500 to 600%. In another embodiment, antibody production by the B cell population is increased by about 600 to 700%. In another embodiment, antibody production by the B cell population is increased by about 700 to 800%. In another embodiment, antibody production by the B cell population is increased by about 800 to 900%. In another embodiment, antibody production by the B cell population is increased by about 900 to 1000%. In some embodiments, the method further promotes the formation of memory B cells capable of producing the antibody in response to the antigen.

In some embodiments, provided herein is a method for increasing the secretion of pro-inflammatory cytokines from a population of immune cells comprising allowing the population of immune cells to interact with a polypeptide comprising a single-chain trimeric CD40L fusion protein according to the present disclosure Antigen-presenting cell populations come into contact. In a specific embodiment, the polypeptide contacted with the population of antigen-presenting cells is a single-chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and ( b) Fc monomer peptide. In some embodiments, the population of antigen-presenting cells presents the antigen to the population of immune cells. In some embodiments, the population of immune cells comprises T cells. In some embodiments, the immune cell population is a T cell population. In specific embodiments, upon activation of the antigen-presenting cells, antigen presentation by the antigen-presenting cell population of the immune cell population is increased. In some embodiments, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In one embodiment, the minimal percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 10 to 20%. In another embodiment, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 20 to 30%. In another embodiment, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 30 to 40%. In another embodiment, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 40 to 50%. In another embodiment, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 50 to 60%. In another embodiment, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 60 to 70%. In another embodiment, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 70 to 80%. In another embodiment, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 80 to 90%. In another embodiment, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 90 to 99%. In some embodiments, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 10%. In some embodiments, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 20%. In some embodiments, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 30%. In some embodiments, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 40%. In some embodiments, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 45%. In some embodiments, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 50%. In some embodiments, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 55%. In some embodiments, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 60%. In some embodiments, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 65%. In some embodiments, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 70%. In some embodiments, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 75%. In some embodiments, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 80%. In some embodiments, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 85%. In some embodiments, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 90%. In some embodiments, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 95%. In some embodiments, the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about or 99%. In some embodiments, the population of antigen-presenting cells comprises B cells, dendritic cells, macrophages, natural killer cells, monocytes, granulocytes, eosinophils, neutrophils, or combinations thereof. In some embodiments, the population of antigen presenting cells comprises B cells. In some embodiments, the population of antigen presenting cells comprises macrophages. In some embodiments, the population of antigen presenting cells comprises dendritic cells. In some embodiments, the population of antigen presenting cells comprises natural killer cells. In some embodiments, the population of antigen presenting cells comprises monocytes. In some embodiments, the population of antigen presenting cells comprises spheroids. In some embodiments, the population of antigen-presenting cells comprises eosinophils. In some embodiments, the population of antigen presenting cells comprises neutrophils. In some embodiments, the cytokines are IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, or any combination thereof. In one embodiment, the interleukin is IL-1. In one embodiment, the interleukin is IL-2. In one embodiment, the interleukin is IL-6. In one embodiment, the interleukin is IL-12. In one embodiment, the interleukin is IL-17. In one embodiment, the interleukin is IL-22. In one embodiment, the interleukin is IL-23. In one embodiment, the cytokine is GM-CSF. In one embodiment, the cytokine is TNF-α. In one embodiment, the cytokine is IFN-γ. In some embodiments, interleukin production is increased by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% %, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% . In one embodiment, interleukin production is increased by about 10 to 100%. In another embodiment, interleukin production is increased by about 100 to 200%. In another embodiment, interleukin production is increased by about 200 to 300%. In another embodiment, interleukin production is increased by about 300 to 400%. In another embodiment, interleukin production is increased by about 400 to 500%. In another embodiment, interleukin production is increased by about 500 to 600%. In another embodiment, interleukin production is increased by about 600 to 700%. In another embodiment, interleukin production is increased by about 700 to 800%. In another embodiment, interleukin production is increased by about 800 to 900%. In another embodiment, interleukin production is increased by about 900 to 1000%. In another embodiment, interleukin production is increased by about 10 to 1000%. In one embodiment, interleukin production is increased by about 20%. In one embodiment, interleukin production is increased by about 30%. In one embodiment, interleukin production is increased by about 40%. In one embodiment, interleukin production is increased by about 45%. In one embodiment, interleukin production is increased by about 50%. In one embodiment, cytokine production is increased by about 55%. In one embodiment, interleukin production is increased by about 60%. In one embodiment, cytokine production is increased by about 65%. In one embodiment, cytokine production is increased by about 70%. In one embodiment, cytokine production is increased by about 75%. In one embodiment, cytokine production is increased by about 80%. In one embodiment, cytokine production is increased by about 85%. In one embodiment, cytokine production is increased by about 90%. In one embodiment, cytokine production is increased by about 95%. In one embodiment, interleukin production is increased by about 100%. In one embodiment, interleukin production is increased by about 125%. In one embodiment, interleukin production is increased by about 150%. In one embodiment, interleukin production is increased by about 175%. In one embodiment, cytokine production is increased by about 200%. In one embodiment, cytokine production is increased by about 250%. In one embodiment, cytokine production is increased by about 300%. In one embodiment, cytokine production is increased by about 400%. In one embodiment, cytokine production is increased by about 500%. In one embodiment, cytokine production is increased by about 600%. In one embodiment, cytokine production is increased by about 700%. In one embodiment, cytokine production is increased by about 800%. In one embodiment, cytokine production is increased by about 900%. In one embodiment, interleukin production is increased by about 1000%.

In some embodiments, provided herein is a method for increasing the phagocytosis of diseased cells by a population of macrophages, comprising allowing the diseased cells, the macrophages, or both the diseased cells and the macrophages to were contacted with a polypeptide comprising a single-chain trimeric CD40L fusion protein according to the present disclosure. In a specific embodiment, the polypeptide that contacts a population of macrophages is a single chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and ( b) Fc monomer peptide.

In some embodiments, the minimum percentage of phagocytic macrophages in the population of macrophages is increased by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70% %, 75%, 80%, 85%, 90%, 95%, or 99%. In one embodiment, the minimal percentage of phagocytic macrophages in the macrophage population is increased by about 10 to 20%. In another embodiment, the minimal percentage of phagocytic macrophages in the population of macrophages is increased by about 20 to 30%. In another embodiment, the minimal percentage of phagocytic macrophages in the macrophage population is increased by about 30 to 40%. In another embodiment, the minimal percentage of phagocytic macrophages in the macrophage population is increased by about 40 to 50%. In another embodiment, the minimal percentage of phagocytic macrophages in the macrophage population is increased by about 50 to 60%. In another embodiment, the minimal percentage of phagocytic macrophages in the population of macrophages is increased by about 60 to 70%. In another embodiment, the minimal percentage of phagocytic macrophages in the population of macrophages is increased by about 70 to 80%. In another embodiment, the minimal percentage of phagocytic macrophages in the population of macrophages is increased by about 80 to 90%. In another embodiment, the minimal percentage of phagocytic macrophages in the population of macrophages is increased by about 90 to 99%. In some embodiments, the minimum percentage of phagocytic macrophages in the macrophage population is increased by about 10%. In some embodiments, the minimum percentage of phagocytic macrophages in the macrophage population is increased by about 20%. In some embodiments, the minimum percentage of phagocytic macrophages in the population of macrophages is increased by about 30%. In some embodiments, the minimum percentage of phagocytic macrophages in the macrophage population is increased by about 40%. In some embodiments, the minimum percentage of phagocytic macrophages in the macrophage population is increased by about 45%. In some embodiments, the minimum percentage of phagocytic macrophages in the population of macrophages is increased by about 50%. In some embodiments, the minimum percentage of phagocytic macrophages in the macrophage population is increased by about 55%. In some embodiments, the minimum percentage of phagocytic macrophages in the macrophage population is increased by about 60%. In some embodiments, the minimum percentage of phagocytic macrophages in the population of macrophages is increased by about 65%. In some embodiments, the minimal percentage of phagocytic macrophages in the population of macrophages is increased by about 70%. In some embodiments, the minimal percentage of phagocytic macrophages in the population of macrophages is increased by about 75%. In some embodiments, the minimum percentage of phagocytic macrophages in the population of macrophages is increased by about 80%. In some embodiments, the minimal percentage of phagocytic macrophages in the population of macrophages is increased by about 85%. In some embodiments, the minimal percentage of phagocytic macrophages in the population of macrophages is increased by about 90%. In some embodiments, the minimal percentage of phagocytic macrophages in the population of macrophages is increased by about 95%. In some embodiments, the minimal percentage of phagocytic macrophages in the population of macrophages is increased by about or 99%.

In some embodiments, the phagocytosis of macrophages is measured by co-culturing macrophages labeled with a first fluorescent dye with diseased cells labeled with a second fluorescent dye, wherein the first The fluorescent dye is different from the second fluorescent dye. In some embodiments, the percentage of phagocytic macrophages is measured by determining the percentage of macrophages comprising the diseased cells. In a specific embodiment, the diseased cell is a cancer cell. In specific embodiments, the diseased cell line is a cell infected with an infectious pathogen. In some embodiments, the infection pathogenic system is a virus, bacterium, fungus, or parasite.

In some embodiments, provided herein is a method for increasing antigen presentation in a population of dendritic cells comprising contacting the dendritic cells with a polypeptide comprising a single-chain trimeric CD40L fusion protein according to the disclosure. In a specific embodiment, the polypeptide contacting a population of dendritic cells is a single chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and ( b) Fc monomer peptide. In some embodiments, the antigen is derived from or derived from an infectious pathogen. In some embodiments, the antigen is derived from or derived from a diseased cell. In some embodiments, the diseased cell is a cancer cell. In some embodiments, the diseased cell line is a cell infected with an infectious pathogen. In some embodiments, the infectious agent is a virus, bacterium, fungus, parasite, or a combination thereof. In some embodiments, the antigen is chemically conjugated or recombinantly fused to a polypeptide comprising a single-chain trimeric CD40L fusion protein.

In some embodiments, the antigen presentation by the dendritic cells is measured by co-culturing dendritic cells labeled with a first fluorescent dye with the antigen labeled with a second fluorescent dye, wherein The first fluorescent dye is different from the second fluorescent dye. In some embodiments, the percentage of dendritic cells presenting the antigen is measured by determining the percentage of dendritic cells in the population of dendritic cells that colocalize with the antigen. In some embodiments, the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. In one embodiment, the minimal percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 10 to 20%. In another embodiment, the minimal percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 20 to 30%. In another embodiment, the minimal percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 30 to 40%. In another embodiment, the minimal percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 40 to 50%. In another embodiment, the minimal percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 50 to 60%. In another embodiment, the minimal percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 60 to 70%. In another embodiment, the minimal percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 70 to 80%. In another embodiment, the minimal percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 80 to 90%. In another embodiment, the minimal percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 90 to 99%. In some embodiments, the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 10%. In some embodiments, the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 20%. In some embodiments, the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 30%. In some embodiments, the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 40%. In some embodiments, the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 45%. In some embodiments, the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 50%. In some embodiments, the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 55%. In some embodiments, the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 60%. In some embodiments, the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 65%. In some embodiments, the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 70%. In some embodiments, the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 75%. In some embodiments, the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 80%. In some embodiments, the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 85%. In some embodiments, the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 90%. In some embodiments, the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 95%. In some embodiments, the minimal percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about or 99%.

In some embodiments, provided herein is a method for increasing expression of a CD40 polypeptide by a target cell comprising contacting the target cell with a polypeptide comprising a single-chain trimeric CD40L fusion protein according to the disclosure. In a specific embodiment, the polypeptide in contact with the target cell is a single chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and (b) Fc monomer peptide.

In some embodiments, the target cell is a diseased cell. In some embodiments, the diseased cell is a cancer cell. In some embodiments, the diseased cell line is a cell infected with an infectious pathogen. In some embodiments, the infectious agent is a virus, bacterium, fungus, parasite, or a combination thereof. In some embodiments, the target cell is an immune cell selected from the group consisting of B cells, natural killer cells, dendritic cells, macrophages, monocytes, granulocytes, eosinophils, neutrophils, or combination.

In some embodiments, the diseased cell population is reduced by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70% %, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In one embodiment, the diseased cell population is reduced by about 10 to 20%. In another embodiment, the diseased cell population is reduced by about 20 to 30%. In another embodiment, the diseased cell population is reduced by about 30 to 40%. In another embodiment, the diseased cell population is reduced by about 40 to 50%. In another embodiment, the diseased cell population is reduced by about 50 to 60%. In another embodiment, the diseased cell population is reduced by about 60 to 70%. In another embodiment, the diseased cell population is reduced by about 70 to 80%. In another embodiment, the diseased cell population is reduced by about 80 to 90%. In another embodiment, the diseased cell population is reduced by about 90 to 99%. In one embodiment, the diseased cell population is reduced by about 10%. In one embodiment, the diseased cell population is reduced by about 20%. In one embodiment, the diseased cell population is reduced by about 30%. In one embodiment, the diseased cell population is reduced by about 40%. In one embodiment, the diseased cell population is reduced by about 45%. In one embodiment, the diseased cell population is reduced by about 50%. In one embodiment, the diseased cell population is reduced by about 55%. In one embodiment, the diseased cell population is reduced by about 60%. In one embodiment, the diseased cell population is reduced by about 65%. In one embodiment, the diseased cell population is reduced by about 70%. In one embodiment, the diseased cell population is reduced by about 75%. In one embodiment, the diseased cell population is reduced by about 80%. In one embodiment, the diseased cell population is reduced by about 85%. In one embodiment, the diseased cell population is reduced by about 90%. In one embodiment, the diseased cell population is reduced by about 95%. In one embodiment, the diseased cell population is reduced by about 99%. In one embodiment, the diseased cell population is reduced by about 100%.

In some embodiments, provided herein is a method for creating a pro-inflammatory environment in tissue surrounding a population of diseased cells comprising contacting the tissue with a polypeptide comprising a single-chain trimeric CD40L fusion protein according to the disclosure. In a specific embodiment, the tissue-contacting polypeptide is a single-chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and (b) an Fc monomeric peptides.

In some embodiments, activated B cells, CD4+ T cells, CD8+ T cells, MAIT, dendritic cells, macrophages, natural killer cells, monocytes, granulocytes, eosinophils, and/or Increased infiltration of neutrophils. In some embodiments, the infiltration of activated B cells is increased. In some embodiments, infiltration of CD4+ T cells is increased. In some embodiments, infiltration of CD8+ T cells is increased. In some embodiments, the infiltration of dendritic cells is increased. In some embodiments, macrophage infiltration is increased. In some embodiments, natural killer cell infiltration is increased. In some embodiments, the infiltration of mononuclear spheres is increased. In some embodiments, the infiltration system of the particle spheres is increased. In some embodiments, the infiltration of eosinophilic spheres is increased. In some embodiments, the infiltration of neutrophils is increased.

In some embodiments, the concentration of pro-inflammatory cytokines is increased in the tissue. In some embodiments, the pro-inflammatory cytokines are IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN- γ, or any combination thereof. In one embodiment, the interleukin is IL-1. In one embodiment, the interleukin is IL-2. In one embodiment, the interleukin is IL-6. In one embodiment, the interleukin is IL-12. In one embodiment, the interleukin is IL-17. In one embodiment, the interleukin is IL-22. In one embodiment, the interleukin is IL-23. In one embodiment, the cytokine is GM-CSF. In one embodiment, the cytokine is TNF-α. In one embodiment, the cytokine is IFN-γ.

In some embodiments, antigen presentation by antigen presenting cells from or derived from the diseased cells is increased in the tissue. In some embodiments, phagocytosis of the diseased cells is increased in the tissue. In some embodiments, apoptosis of the diseased cells caused by cell-mediated cytotoxicity is increased in the tissue. In some embodiments, apoptosis of the diseased cells caused by antibody-dependent cellular cytotoxicity is increased in the tissue. In some embodiments, the diseased cell population is reduced in the tissue. In some embodiments, the diseased cell population is reduced by about 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 95%, or 99%. In one embodiment, the diseased cell population is reduced by about 10 to 20%. In another embodiment, the diseased cell population is reduced by about 20 to 30%. In another embodiment, the diseased cell population is reduced by about 30 to 40%. In another embodiment, the diseased cell population is reduced by about 40 to 50%. In another embodiment, the diseased cell population is reduced by about 50 to 60%. In another embodiment, the diseased cell population is reduced by about 60 to 70%. In another embodiment, the diseased cell population is reduced by about 70 to 80%. In another embodiment, the diseased cell population is reduced by about 80 to 90%. In another embodiment, the diseased cell population is reduced by about 90 to 99%. In one embodiment, the diseased cell population is reduced by about 10%. In one embodiment, the diseased cell population is reduced by about 20%. In one embodiment, the diseased cell population is reduced by about 30%. In one embodiment, the diseased cell population is reduced by about 40%. In one embodiment, the diseased cell population is reduced by about 45%. In one embodiment, the diseased cell population is reduced by about 50%. In one embodiment, the diseased cell population is reduced by about 55%. In one embodiment, the diseased cell population is reduced by about 60%. In one embodiment, the diseased cell population is reduced by about 65%. In one embodiment, the diseased cell population is reduced by about 70%. In one embodiment, the diseased cell population is reduced by about 75%. In one embodiment, the diseased cell population is reduced by about 80%. In one embodiment, the diseased cell population is reduced by about 85%. In one embodiment, the diseased cell population is reduced by about 90%. In one embodiment, the diseased cell population is reduced by about 95%. In one embodiment, the diseased cell population is reduced by about 99%. In one embodiment, the diseased cell population is reduced by about 100%. Instructions

According to certain embodiments, the single-chain trimeric CD40L Fc fusion protein may be provided in a buffered composition for storage or use. A suitable buffer for storing the single-chain trimeric CD40L Fc fusion protein will be used to maintain

In one aspect, provided herein is a method of treating a disease.

Also provided herein is a single chain trimeric CD40L Fc fusion protein as described herein for use in therapy. Such therapy includes methods of treatment and uses for treatment as defined herein and all embodiments thereof.

Also provided herein is a single chain trimeric CD40L Fc fusion protein as described herein for use in the treatment of a disease or disorder. Such methods include methods of treatment of diseases or conditions according to the definitions herein and all embodiments thereof.

In some embodiments, provided herein is a method for eliminating diseased cells in a subject comprising administering to the subject an effective amount of a polypeptide comprising a single-chain trimeric CD40L fusion protein according to the present disclosure. In specific embodiments, the polypeptide administered to a subject is a single chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and (b) Fc monomer peptide. In some embodiments, the diseased cell does not express a CD40 polypeptide. In some embodiments, the diseased cell expresses a CD40 polypeptide. In some embodiments, the diseased cell is a cancer cell. In some embodiments, the diseased cell line is a cell infected with an infectious pathogen. In some embodiments, the infectious agent is a virus, bacterium, fungus, parasite, or a combination thereof.

Also provided herein is a single chain trimeric CD40L Fc fusion protein as described herein for use in eliminating diseased cells in a subject. In certain embodiments, the diseased cell is a cancer cell. In other embodiments, the diseased cell is a cell infected with a pathogen.

In some embodiments, provided herein is a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a polypeptide comprising a single-chain trimeric CD40L fusion protein according to the present disclosure. In specific embodiments, the polypeptide administered to a subject is a single chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and (b) Fc monomer peptide.

Also provided is a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a dimer comprising two single-chain trimeric CD40L Fc fusion proteins each comprising: (a) three CD40L subunits, which are covalently linked to each other by a peptide linker; and (b) an Fc monomer peptide. In some embodiments, the methods comprise administering a homodimer. In some embodiments, the methods comprise administering a homodimer.

Also provided herein is a polypeptide comprising a single-chain trimeric CD40L fusion protein as described herein for use in the treatment of cancer. Such treatment includes those according to the method of treating cancer as defined herein and all embodiments thereof. In a specific embodiment, the polypeptide for treating cancer is a single chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and (b) Fc monomer peptide.

Also provided herein is a single chain trimeric CD40L Fc fusion protein as described herein for use in the treatment of cancer. Such treatment includes those according to the method of treating cancer as defined herein and all embodiments thereof.

Also provided herein is a dimer comprising two single chain trimeric CD40L Fc fusion proteins as described herein for use in the treatment of cancer. Such treatment includes those according to the method of treating cancer as defined herein and all embodiments thereof.

In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is liquid cancer. In some embodiments, the cancer is selected from the group consisting of melanoma, mesothelioma, advanced solid tumors, and lymphoma. In one embodiment, the cancer is melanoma. In one embodiment, the cancer is mesothelioma. In one embodiment, the cancer is an advanced solid tumor. In one embodiment, the cancer is lymphoma.

In some embodiments, the method comprises administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a single chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits , which are covalently linked to each other by a peptide linker; and (b) Fc monomeric peptides. In some embodiments, the methods comprise administering a homodimer. In some embodiments, the methods comprise administering a heterodimer.

In some embodiments, the method of treating cancer enhances the innate anti-neoplastic immune response. As used herein, "anti-neoplastic immune response" refers to the recruitment, priming, and/or activation of an individual's immune cells (such as, for example, T cells, B cells) To launch an immune response against a specific tumor target. In some embodiments, antigen-presenting cells activated by polypeptides comprising single-chain trimeric CD40L fusion proteins according to the present disclosure may in turn activate additional cell types, such as T cells. Replacement Typically, an antitumor immune response includes a reduction in tumor burden. The term also encompasses the recruitment of cytokines, such as granulocyte macrophage colony stimulating factor (GM-CSF), which serve to amplify immune activation And/or induce migration of stimulated cells to lymph nodes.

In one embodiment, the disease is cancer. In some embodiments, the cancer is selected from the group consisting of melanoma, mesothelioma, advanced solid tumors, and lymphoma. In some embodiments, the cancer is a solid tumor cancer. In other embodiments, the cancer is a blood cancer.

In some embodiments, the cancer is adrenal cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, esophagus cancer, gallbladder cancer, gestational trophoblast, head and neck cancer Carcinoma, Hodgkin's lymphoma, bowel cancer, kidney cancer, leukemia, liver cancer, lung cancer, melanoma, mesothelioma, multiple myeloma, neuroendocrine tumors, non-Hodgkin's lymphoma, oral cancer, ovarian cancer , pancreatic cancer, prostate cancer, sinus cancer, skin cancer, soft tissue sarcoma, spine cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, uterine cancer, endometrial cancer, vaginal cancer, or vulvar cancer.

In some embodiments, the adrenal cancer is adrenocortical carcinoma (ACC), adrenocortical carcinoma, pheochromocytoma, or neuroblastoma. In some embodiments, the anal cancer is squamous cell carcinoma, cloacal carcinoma, adenocarcinoma, basal cell carcinoma, or melanoma. In some embodiments, the cancer of the appendix is a neuroendocrine tumor (NET), mucinous adenocarcinoma, goblet cell carcinoid, intestinal adenocarcinoma, or ring cell adenocarcinoma. In some embodiments, the cholangiocarcinoma is extrahepatic cholangiocarcinoma, adenocarcinoma, hilar cholangiocarcinoma, perihilar cholangiocarcinoma, distal cholangiocarcinoma, or intrahepatic cholangiocarcinoma. In some embodiments, the bladder cancer is transitional cell carcinoma (TCC), papillary carcinoma, squamous carcinoma, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, or sarcoma. In some embodiments, the bone cancer is primary bone cancer, sarcoma, osteosarcoma, chondrosarcoma, sarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma, or metastatic bone cancer. In some embodiments, the brain cancer is astrocytoma, brainstem glioma, glioblastoma, meningioma, ependymoma, oligodendritic glioma, mixed glioma, encephalopathy Body cancer, pituitary adenoma, craniopharyngioma, germ cell tumor, pineal region tumor, medulloblastoma, or primary CNS lymphoma. In some embodiments, the breast cancer is breast adenocarcinoma, invasive breast cancer, non-invasive breast cancer, breast sarcoma, histologically deforming carcinoma, adenocystic carcinoma, phyllodes tumor, angiosarcoma, HER2-positive breast cancer, triple-negative breast cancer, or Inflammatory breast cancer. In some embodiments, the cervical cancer is squamous cell carcinoma, or adenocarcinoma. In some embodiments, the colorectal cancer is colorectal adenocarcinoma, primary colorectal lymphoma, gastrointestinal stromal tumor, leiomyosarcoma, carcinoid tumor, mucinous adenocarcinoma, ring cell adenocarcinoma, gastrointestinal carcinoid tumor , or melanoma. In some embodiments, the esophageal cancer is adenocarcinoma, or squamous cell carcinoma. In some embodiments, the gallbladder cancer is adenocarcinoma, papillary adenocarcinoma, adenosquamous carcinoma, squamous cell carcinoma, small cell carcinoma, or sarcoma. In some embodiments, the gestational trophoblastic disease (GTD) is mole, gestational trophoblastic tumor (GTN), choriocarcinoma, placental site trophoblastic tumor (PSTT), or epithelioid trophoblastic tumor (ETT). In some embodiments, the head and neck cancer is cancer of the larynx, nasopharynx, hypopharynx, nasal cavity, paranasal sinuses, salivary glands, oral cavity, oropharynx, or tonsils. In some embodiments, the Hodgkin's lymphoma is classical Hodgkin's lymphoma, tuberous sclerosis, mixed cellularity, lymphocyte-rich, lymphocyte-depleted, or nodular lymphocyte-predominant Hodgkin's lymphoma King's Lymphoma (NLPHL). In some embodiments, the bowel cancer is small bowel cancer, small bowel cancer, adenocarcinoma, sarcoma, gastrointestinal stromal tumor, carcinoid tumor, or lymphoma. In some embodiments, the kidney cancer is renal cell carcinoma (RCC), clear cell RCC, papillary RCC, chromophobe RCC, collecting duct RCC, unclassified RCC, transitional cell carcinoma, urinary tract carcinoma, renal pelvis carcinoma, or renal cell carcinoma sarcoma. In some embodiments, the leukemia is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), Or myelodysplastic syndrome (MDS). In a specific embodiment, the leukemia is AML. In some embodiments, the liver cancer is hepatocellular carcinoma (HCC), fibrolamellar HCC, cholangiocarcinoma, angiosarcoma, or liver metastasis. In some embodiments, the lung cancer is small cell lung cancer, small cell carcinoma, combined small cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung cancer, large cell undifferentiated carcinoma, pulmonary nodule, metastatic lung cancer, adenocarcinoma Squamous cell carcinoma, large cell neuroendocrine carcinoma, salivary gland lung carcinoma, lung carcinoid, mesothelioma, lung sarcomatoid carcinoma, or malignant granulosa cell lung neoplasm. In some embodiments, the melanoma is superficial spreading melanoma, nodular melanoma, acral small nevus melanoma, small nevus malignant melanoma, amelanoma melanoma, desmoplastic melanoma, ocular Melanoma, or metastatic melanoma. In some embodiments, the mesothelioma is pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, or testicular mesothelioma. In some embodiments, the multiple myeloma is active myeloma, or smoldering myeloma. In some embodiments, the neuroendocrine tumor is a neuroendocrine tumor of the gastrointestinal tract, a neuroendocrine tumor of the pancreas, or a neuroendocrine tumor of the lung. In some embodiments, the non-Hodgkin's lymphoma is anaplastic large cell lymphoma, lymphoblastic lymphoma, peripheral T-cell lymphoma, follicular lymphoma, cutaneous T-cell lymphoma, lymphoplasmacytic Lymphoma, marginal zone B-cell lymphoma, MALT lymphoma, small cell lymphocytic lymphoma, Burkitt's lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), prodromal T Lymphoblastic leukemia/lymphoma, acute lymphoblastic leukemia (ALL), adult T-cell lymphoma/leukemia (ATLL), hairy cell leukemia, B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Primary mediastinal B-cell lymphoma, primary central nervous system (CNS) lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, nodular marginal zone B Cell Lymphoma, Splenic Marginal Zone B-Cell Lymphoma, Lymphoplasmacytic Lymphoma, B-Cell Non-Hodgkin's Lymphoma, T-Cell Non-Hodgkin's Lymphoma, Natural Killer Cell Lymphoma, Cutaneous T-Cell Lymphoma , Alibert-Bazin syndrome, Sezary syndrome, primary cutaneous degenerative large cell lymphoma, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), systemic ALCL, enteropathic T-cell lymphoma (EATL), or hepatosplenic gamma/delta T-cell lymphoma. In some embodiments, the oral cancer is squamous cell carcinoma, verrucous carcinoma, minor salivary gland carcinoma, lymphoma, benign oral tumor, eosinophilic granuloma, fibroma, granulosa cell tumor, keratoacanthoma, leiomyoma, bone Enchondroma, lipoma, schwannoma, neurofibroma, papilloma, condyloma acuminatum, verrucous xanthoma, pyrogenic granuloma, rhabdomyoma, odontogenic neoplasm, leukoplakia, erythema of mucosa, squamous cell lip carcinoma of the lip, basal cell lip, mouth, gums, or tongue. In some embodiments, the ovarian cancer is epithelial ovarian cancer, mucinous epithelial ovarian cancer, endometrioid epithelial ovarian cancer, clear cell epithelial ovarian cancer, undifferentiated epithelial ovarian cancer, low grade ovarian tumor, primary peritoneal cancer , fallopian tube carcinoma, germ cell tumor, teratoma, asexual embryooma ovarian germ cell carcinoma, endodermal sinus tumor, sex cord stromal tumor, sex cord gonadal stromal tumor, ovarian stromal tumor, granulosa cell tumor, granulosa cell and Sheath cell tumor, Sertoli-Leydig's tumor, ovarian sarcoma, ovarian carcinosarcoma, ovarian adenosarcoma, ovarian leiomyosarcoma, ovarian fibrosarcoma, Krukenberg's tumor, or ovarian cyst. In some embodiments, the pancreatic cancer is exocrine pancreatic adenocarcinoma, pancreatic endocrine adenocarcinoma, or pancreatic cancer, islet cell tumor, or neuroendocrine tumor. In some embodiments, the prostate cancer is prostate adenocarcinoma, prostate sarcoma, transitional cell carcinoma, small cell carcinoma, or neuroendocrine tumor. In some embodiments, the sinus cancer is squamous cell carcinoma, mucosal cell carcinoma, adenoid cystic cell carcinoma, acinar cell carcinoma, sinonasal undifferentiated carcinoma, nasal cavity carcinoma, paranasal sinus carcinoma, maxillary sinus carcinoma, ethmoid sinus carcinoma , or nasopharyngeal carcinoma. In some embodiments, the skin cancer is basal cell carcinoma, squamous cell carcinoma, melanoma, Merkel's cell carcinoma, Kaposi's sarcoma (KS), actinic keratosis, cutaneous lymphoma, or keratoacanthoma. In some embodiments, the soft tissue cancer is angiosarcoma, dermatofibrosarcoma, epithelioid sarcoma, Ewing's sarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), Kaposi's sarcoma, leiomyosarcoma, liposarcoma, dedifferentiated liposarcoma (DL), myxoid/round cell liposarcoma (MRCL), well-differentiated liposarcoma (WDL), malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma (RMS), or synovial sarcoma. In some embodiments, the spinal cancer is a spinal metastatic tumor. In some embodiments, the gastric cancer is gastric adenocarcinoma, gastric lymphoma, gastrointestinal stromal tumor, carcinoid tumor, gastric carcinoid tumor, type I ECL cell carcinoid, type II ECL cell carcinoid, or type III ECL cell cell carcinoid. In some embodiments, the testicular cancer is a seminoma, nonseminoma, embryonal carcinoma, yolk sac carcinoma, choriocarcinoma, teratoma, gonadal stromal tumor, Leydig's cell tumor, or Sertoli's cell tumor. In some embodiments, the laryngeal cancer is squamous cell carcinoma, adenocarcinoma, sarcoma, laryngeal carcinoma, pharyngeal carcinoma, nasopharyngeal carcinoma, oropharyngeal carcinoma, hypopharyngeal carcinoma, laryngeal carcinoma, laryngeal squamous cell carcinoma, laryngeal adenocarcinoma, Lymphoepithelioma, spindle cell carcinoma, verrucous carcinoma, anaplastic carcinoma, or lymph node carcinoma. In some embodiments, the thyroid cancer is papillary carcinoma, follicular carcinoma, Hürthle cell carcinoma, medullary thyroid carcinoma, or degenerative carcinoma. In some embodiments, the uterine cancer is endometrial carcinoma, endometrial adenocarcinoma, endometrioid carcinoma, serous adenocarcinoma, adenosquamous carcinoma, uterine carcinosarcoma, uterine sarcoma, uterine leiomyosarcoma, endometrial stromal sarcoma , or undifferentiated sarcoma. In some embodiments, the vaginal cancer is squamous cell carcinoma, adenocarcinoma, melanoma, or sarcoma. In some embodiments, the vulvar cancer is squamous cell carcinoma, or adenocarcinoma.

In one aspect, an object is an object of need. In another aspect, the subject is a human being.

As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of an active ingredient or component that elicits a desired biological or medical response in a subject. The term means sufficient to treat, diagnose, prevent, and/or treat, diagnose, prevent, and/or treat symptom(s) of a disease, disorder, and/or condition when administered to a subject suffering from or susceptible to the disease, disorder, and/or condition Amount to delay onset. Those of ordinary skill in the art will appreciate that a therapeutically effective amount is generally administered via a dosage regimen comprising at least one unit dose.

According to specific embodiments, a therapeutically effective amount refers to an amount sufficient to achieve one or more, two or more, three or more, four or more, or five or more of the following effects: (i) reducing or ameliorate the severity of the disease, disorder, or condition being treated, or symptoms associated therewith; (ii) reduce the duration of the disease, disorder, or condition being treated, or symptoms associated therewith; (iii) prevent the disease being treated , disease, or condition, or progression of symptoms associated therewith; (iv) causing regression of the disease, disease, or condition to be treated, or symptoms associated therewith; (v) preventing or being associated with the disease, disease, or condition to be treated (vi) preventing recurrence of the disease, disorder, or condition being treated or symptoms associated therewith; (vii) reducing the number of subjects suffering from or having symptoms associated with the disease, disorder, or condition being treated (viii) reducing the length of hospitalization of subjects suffering from or having symptoms associated with the disease, disorder, or condition being treated; (ix) increasing the length of hospitalization of subjects suffering from or having symptoms associated with the disease, disorder, or condition being treated survival of the subject; (xi) inhibiting or reducing the disease, disorder, or condition being treated in the subject or symptoms associated therewith; and/or (xii) enhancing or improving the disease preventive or therapeutic effect(s) of another therapy.

A therapeutically effective amount or dosage can vary depending on various factors, such as the disease, disorder or condition to be treated, the means of administration, the target site, the physiological state of the subject (including, for example, age, weight, health), whether the subject is human or animal, Other administrations of drugs, and treatments are either disease prophylactic or therapeutic. Treatment doses are optimally titrated to optimize safety and efficacy.

According to particular embodiments, the compositions described herein are formulated for the intended route of administration to the subject. For example, the compositions described herein can be formulated for intravenous, subcutaneous, or intramuscular administration.

As used herein, the terms "treat, treating, and treatment" are all intended to refer to the improvement or reversal of at least one measurable physical parameter associated with cancer, which may not necessarily be detectable in the subject, but may be tied to the subject. perceptible in. The term "treating" can also refer to causing regression of a disease, disorder, or condition, preventing the progression of a disease, disorder, or condition, or at least delaying the progression of a disease, disorder, or condition. In one embodiment, "treating" means alleviating one or more symptoms associated with a disease, disorder, or condition, preventing the development or onset of one or more symptoms associated with a disease, disorder, or condition, or reducing The duration of one or more symptoms associated with a disease, disorder, or condition, such as a tumor or, more preferably, cancer. In one embodiment, "treating" refers to preventing recurrence of a disease, disorder, or condition. In one embodiment, "treating" refers to increasing the survival of a subject with a disease, disorder, or condition. In one embodiment, "treating" refers to excluding a disease, disorder, or condition in a subject.

In some embodiments, a single chain trimeric CD40L Fc fusion protein or fragment thereof as provided herein is used in combination with a second therapy. In some embodiments, the second therapy is selected from the group consisting of surgery, radiation therapy, chemotherapy, immunotherapy, targeted therapy, hormonal therapy, bone marrow transplantation, cryoablation, and radiofrequency ablation.

In some embodiments, the second therapy is immunotherapy. In some embodiments, immunotherapy encompasses monoclonal antibodies, ad hoc tumor treatments such as checkpoint inhibitors, T cell therapy such as chimeric antigen receptor (CAR) T cell therapy, or cancer vaccines.

As used herein, the term "in combination" in the context of administering two or more therapies to a subject refers to the use of more than one therapy. Use of the term "combination" does not limit the order in which the therapies are administered to a subject. For example, a first therapy (e.g., a composition described herein) can be administered to a subject prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), at the same time or after ( For example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 hours weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks later).

In another aspect, provided herein is a method for treating an infection in a subject in need thereof comprising administering to the subject an effective amount of a polypeptide comprising a single-chain trimeric CD40L fusion protein according to the present disclosure. In specific embodiments, the polypeptide administered to a subject is a single chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and (b) Fc monomer peptide. In some embodiments, the treatment enhances the innate, humoral, or cell-mediated immune response against infection. In one embodiment, the treatment enhances the innate anti-tumor immune response. In one embodiment, the treatment enhances the humoral anti-infective immune response. In one embodiment, the treatment enhances the cell-mediated immune response against infection.

Also provided herein is a polypeptide comprising a single-chain trimeric CD40L fusion protein as described herein for use in the treatment of infection. Such treatment includes those according to the method of treating infection as defined herein and all embodiments thereof. In a specific embodiment, the polypeptide for treating infection is a single chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and (b) Fc monomer peptide.

Also provided herein is a single chain trimeric CD40L Fc fusion protein as described herein for use in the treatment of infection. Such treatment includes those according to the method of treating infection as defined herein and all embodiments thereof.

Also provided herein is a dimer comprising two single chain trimeric CD40L Fc fusion proteins as described herein for use in the treatment of infection. Such treatment includes those according to the method of treating infection as defined herein and all embodiments thereof.

In some embodiments, the infection is caused by an infectious agent such as a virus, bacterium, fungus, or parasite. In one embodiment, the pathogenic system is a virus. In another embodiment, the pathogenic system is bacteria. In one embodiment, the pathogenic system is a fungus. In another embodiment, the pathogenic system is a parasite. In some embodiments, the polypeptide is co-administered with a vaccine composition for preventing the infection in the subject.

In some embodiments, the polypeptide is co-administered simultaneously or sequentially with the vaccine composition.

In another aspect, provided herein is a method for improving a subject's response to a vaccine comprising administering to the subject an effective amount of a polypeptide comprising a single-chain trimeric CD40L fusion protein according to the present disclosure. In a specific embodiment, the single-chain trimeric CD40L fusion protein is administered to the subject simultaneously or sequentially with the vaccine. In specific embodiments, the polypeptide administered to a subject is a single chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and (b) Fc monomer peptide. In some embodiments, the treatment enhances the innate, humoral, or cell-mediated immune response to the vaccine. In one embodiment, the treatment enhances the innate immune response to the vaccine. In one embodiment, the treatment enhances the humoral immune response to the vaccine. In one embodiment, the treatment enhances the cell-mediated immune response to the vaccine.

In some embodiments, vaccines are directed against infectious pathogens, such as viruses, bacteria, fungi, or parasites. In one embodiment, the pathogenic system is a virus. In another embodiment, the pathogenic system is bacteria. In one embodiment, the pathogenic system is a fungus. In another embodiment, the pathogenic system is a parasite. In another embodiment, the vaccine is against cancer. In another embodiment, the vaccine is against tumors. In another embodiment, the vaccine is against an allergen. Other types of vaccines are also contemplated. In some embodiments, the polypeptide is co-administered with a vaccine composition for enhancing a subject's immune response to the vaccine. In some embodiments, polypeptides function as adjuvants for vaccines.

In some embodiments, a single-chain trimeric CD40L Fc fusion protein described herein is administered to a subject in need thereof. In some embodiments, the subject is a human. In yet other embodiments, the single chain trimeric CD40L Fc fusion protein is administered to the subject via oral delivery, buccal delivery, nasal delivery, or inhalation delivery.

In yet other embodiments, provided herein is a use of a single chain trimeric CD40L Fc fusion protein provided herein for treating a disease or disorder in a subject. In some embodiments, therapeutic molecules are administered to a subject via oral delivery. In some embodiments, therapeutic molecules are administered to a subject via buccal delivery. In some embodiments, therapeutic molecules are administered to a subject via nasal delivery. In some embodiments, therapeutic molecules are administered to a subject via inhalational delivery.

Also provided is a system comprising building blocks for providing a single-chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits shared with each other by a peptide linker valence linkage; and (b) Fc monomer peptide.

In another aspect, provided herein is a system comprising a building block for providing a dimer comprising two single-chain trimeric CD40L Fc fusion proteins comprising: (a) three a CD40L subunit covalently linked to each other by a peptide linker; and (b) an Fc monomer peptide.

In yet another aspect, provided herein is a system comprising a means for providing a polynucleotide encoding a single-stranded trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits, which are covalently linked to each other by a peptide linker; and (b) an Fc monomer peptide.

In one aspect, provided herein is a system comprising a building block for providing a vector comprising a polynucleotide encoding a single-stranded trimeric CD40L Fc fusion protein comprising: (a ) three CD40L subunits covalently linked to each other by a peptide linker; and (b) an Fc monomer peptide.

In one aspect, provided herein is a system comprising a building block for providing a vector comprising a polynucleotide encoding a single-stranded trimeric CD40L Fc fusion protein comprising: (a ) three CD40L subunits covalently linked to each other by a peptide linker; and (b) an Fc monomer peptide.

In yet another aspect, provided herein is a system that provides a means for providing a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a single-chain trimeric CD40L comprising Fc fusion protein: (a) three CD40L subunits covalently linked to each other by a peptide linker; and (b) Fc monomeric peptide.

In one aspect, provided herein is a system that provides a kit comprising a single-chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits interconnected by a peptide linker covalently linked; and (b) an Fc monomer peptide.

Also provided are nucleic acid molecules encoding single-chain trimeric CD40L Fc fusion proteins, as well as nucleic acid molecules encoding components of the single-chain trimeric CD40L Fc fusion proteins described herein. Also provided are kits comprising a carrier and packaging for the above. Also provided are host cells comprising vectors containing a nucleic acid molecule encoding a single-chain trimeric CD40L Fc fusion protein described herein.

Also provided is a procedure for making a single-chain trimeric CD40L Fc fusion protein or a fragment thereof.

In one aspect, provided herein is a method for producing a single-stranded trimeric CD40L Fc fusion protein or fragment thereof comprising: (a) introducing into a host cell a polynucleotide encoding a single-stranded trimeric CD40L An Fc fusion protein comprising (i) three CD40L subunits covalently linked to each other by a peptide linker; and (ii) an Fc monomeric peptide; (b) ) cultivating the host cell under conditions to produce the single-chain trimeric CD40L Fc fusion protein or fragment thereof; and (c) recovering the single-chain trimeric CD40L Fc fusion protein or fragment thereof from the cell or culture.

In another aspect, provided herein is a method of producing a dimer comprising two single-stranded trimeric CD40L Fc fusion proteins, the method comprising: (a) introducing a polynucleotide into a host cell, the multinuclear The nucleotides encode a single-chain trimeric CD40L Fc fusion protein comprising (i) three CD40L subunits covalently linked to each other by a peptide linker; and (ii) ) an Fc monomeric peptide; (b) culturing the host cell under conditions to produce the single-chain trimeric CD40L Fc fusion protein or a fragment thereof; (c) recovering the single-chain trimeric CD40L Fc fusion from the cell or culture protein or fragment thereof; and (d) combining a single-chain trimeric CD40L Fc fusion protein or fragment thereof under conditions favorable for dimerization.

In yet another aspect, provided herein is a method of producing a pharmaceutical composition having a single-chain trimeric CD40L Fc fusion protein or a fragment thereof comprising combining the single-chain trimeric CD40L Fc fusion protein or a fragment thereof with a pharmaceutically acceptable The acceptable carrier is combined to obtain the pharmaceutical composition.

In yet another aspect, provided herein is a method of producing a vaccine composition comprising a single-chain trimeric CD40L Fc fusion protein or a fragment thereof, comprising: combining the single-chain trimeric CD40L Fc fusion protein or a fragment thereof with a vaccine antigen combined to obtain the vaccine composition.

In some embodiments, the single-chain trimeric CD40L Fc fusion protein or fragment thereof comprises an IgG-like molecule with complementary CH3 domains that facilitate heterodimerization. In some embodiments, the single-chain trimeric CD40L Fc fusion protein or fragment thereof comprises a recombinant IgG-like dual targeting molecule, wherein the molecule is flanked by at least two Fab fragments of different antibodies or a portion of the Fab fragments. In some embodiments, the single chain trimeric CD40L Fc fusion protein or fragment thereof comprises an IgG fusion molecule wherein a full length IgG antibody is fused to an additional Fab fragment or part of a Fab fragment. In some embodiments, the single-chain trimeric CD40L Fc fusion protein or fragment thereof comprises an Fc fusion molecule, wherein a single-chain Fv molecule or a stabilized bivalent antibody system is fused to a heavy chain constant domain, an Fc region, or a portion thereof. In some embodiments, the single chain trimeric CD40L Fc fusion protein or fragment thereof comprises a Fab fusion molecule wherein different Fab fragments are fused together. In some embodiments, the single chain trimeric CD40L Fc fusion protein or fragment thereof comprises scFv-based and diabody-based heavy chain antibodies (e.g., domain antibodies, nanobodies), wherein different single chain Fv molecules or different bivalent Antibodies or different heavy chain antibodies (eg domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule.

In some embodiments, IgG-like molecules with complementary CH3 domain molecules include Triomab/Quadroma (Trion Pharma/Fresenius Biotech), Knobs-into-Holes (Genentech), CrossMAbs (Roche), and electrostatically-matched ( Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Azymetric™ platform (Zymeworks), and DuoBody (Genmab A/S).

In some embodiments, the recombinant IgG-like dual targeting molecules include dual targeting (Dual Targeting, DT)-Ig (GSK/Domantis), two-in-one Antibody (Genentech), cross-linking ( Cross-linked) mAb (Karmanos Cancer Center), mAb2 (F-Star), and CovX-body (CovX/Pfizer).

In some embodiments, IgG fusion molecules include dual variable domain (Dual Variable Domain, DVD)-Ig (Abbott), class IgG-like Bispecific antibody (IgG-like Bispecific) (InnClone/Eli Lilly), Ts2Ab (MedImmune/ AZ) and BsAb (Zymogenetics), HERCULES (Biogen Idec) and TvAb (Roche).

In some embodiments, Fc fusion molecules may include ScFv/Fc fusion (Academic Institution), SCORPION (Emergent BioSolutions/Trubion, Zymogenetics/BMS), Dual Affinity Retargeting Technology (Fc-DART) (MacroGenics), and double (ScFv) ₂ -Fab (National Research Center for Antibody Medicine--China).

"Homodimerization" as used herein refers to the interaction between two Fc monomeric peptides of the same single chain trimeric CD40L Fc fusion protein. "Homodimer" as used herein refers to molecules with the same single-chain trimeric CD40L Fc fusion protein.

"Heterodimerization" as used herein refers to the interaction between the Fc monomer peptides of a single-chain trimeric CD40L Fc fusion protein with different Fc fusion peptides. "Heterodimer" as used herein refers to a single chain trimeric CD40L Fc fusion protein having two heavy chains with different CH3 amino acid sequences.

A "buttonhole" strategy (see, eg, PCT Publication No. WO2006/028936) can be used to generate a full-length single-chain trimeric CD40L Fc fusion protein. Briefly, selected amino acids that form the CH3 domain interface in human IgG can be mutated at positions that affect CH3 domain interactions to promote heterodimer formation. Amino acids with small side chains (pores) are introduced into the heavy chains of antibodies that specifically bind a first antigen, and amino acids with large side chains (knobs) are introduced into the heavy chains of antibodies that specifically bind a second antigen in the chain. After co-expression of the two antibodies, the heavy chain with the "hole" preferentially interacts with the heavy chain with the "knob" resulting in heterodimer formation. Exemplary CH3 substitution pairs forming a knob and a pore (represented as modified position in the first CH3 domain of the first heavy chain/modified position in the second CH3 domain of the second heavy chain): T366Y/F405A, T366W /F405W, F405W/Y407A, T394W/Y407T, T394S/Y407A, T366W/T394S, F405W/T394S and T366W/T366S_L368A_Y407V.

Other strategies can be used, such as promoting heavy chain heterodimerization using electrostatic interactions that replace positively charged residues on one CH3 surface and negatively charged residues on a second CH3 surface, as described in U.S. Patent Publication No. US2010/0015133 No.; U.S. Patent Publication No. US2009/0182127; U.S. Patent Publication No. US2010/028637; or as described in U.S. Patent Publication No. US2011/0123532. In other strategies, heterodimerization can be promoted by the following substitutions (expressed as modified positions in the first CH3 domain of the first heavy chain/modified positions in the second CH3 domain of the second heavy chain): L351Y_F405AY407V/T394W、T366I_K392M_T394W/F405A_Y407V、T366L_K392M_T394W/F405A_ Y407V、L351Y_Y407A/T366A_K409F、L351Y_Y407A/T366V K409F Y407A/T366A_K409F、或T350V_L351Y_F405A Y407V/T350V_T366L_ K392L_T394W，如美國專利公開案第US2012/0149876號或美國專利公開案第US2013/ as described in No. 0195849.

In certain embodiments, the single chain trimeric CD40L Fc fusion proteins described herein activate CD40 with an _EC50 of less than about 1 pM, less than about 0.9 pM, less than about 0.8 pM, less than about 0.7 pM, less than about 0.6 pM, less than about 0.5 pM, less than about 0.4 pM, less than about 0.300 pM, less than about 0.2 pM, less than about 0.19 pM, less than about 0.18 pM, less than about 0.17 pM, less than about 0.16 pM, less than about 0.15 pM, less than about 0.14 pM, less than about 0.13 pM, less than about 0.12 pM, less than about 0.11 pM, less than about 0.1 pM, less than about 0.09 pM, less than about 0.08 pM, less than about 0.07 pM, less than about 0.06 pM, less than about 0.05 pM, less than about 0.04 pM, less than about 0.03 pM, less than about 0.02 pM, or less than about 0.01 pM. In certain embodiments, the _EC50 is less than about 1000 pM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 300 pM, less than about 200 pM, less than about 190 pM, less than about 180 pM, less than about 170 pM, less than about 160 pM, less than about 150 pM, less than about 140 pM, less than about 130 pM, less than about 120 pM, less than about 110 pM, Less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, or less than about 10 pM .

In certain embodiments, the concentration of the single chain trimeric CD40L Fc fusion protein is about 0.000005 ng/mL, about 0.00005 ng/mL, about 0.0005, about 0.005 ng/mL, about 0.01 ng/mL, about 0.02 ng/mL , about 0.03 ng/mL, about 0.04 ng/mL, about 0.05 ng/mL, about 0.06 ng/mL, about 0.07 ng/mL, about 0.08 ng/mL, about 0.09 ng/mL, about 0.1 ng/mL, about 0.5 ng/mL, ~1.0 ng/mL, ~10 ng/mL, ~20 ng/mL, ~30 ng/mL, ~40 ng/mL, ~50 ng/mL, ~60 ng/mL, ~70 ng /mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, or about 1000 ng/mL.

In some embodiments, one or more components of the single chain trimeric CD40L Fc fusion protein are human. In some embodiments, one or more components of the single chain trimeric CD40L Fc fusion protein are humanized.

In some embodiments, the Fc monomer peptide of the single chain trimeric CD40L Fc fusion protein is derived from an IgG antibody. In some embodiments, the IgG antibody is an IgG1 antibody. In some embodiments, the IgG antibody is an IgG2 antibody. In some embodiments, the IgG antibody is an IgG3 antibody. In some embodiments, the IgG antibody is an IgG4 antibody.

In another general aspect, the invention relates to a vector comprising a polynucleotide encoding a single-stranded trimeric CD40L Fc fusion protein or a fragment thereof. In light of the present disclosure, any vector known to those of ordinary skill in the art, such as plastids, cosmids, phage vectors, or viral vectors, may be used. In some embodiments, the vector is a recombinant expression vector, such as a plastid. The vector may include any element established to perform the conventional functions of the vector, such as promoters, ribosome binding elements, terminators, enhancers, selectable markers, and origins of replication. Promoters can be constitutive, inducible, or repressible. Many expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein to produce antibodies or antigen-binding fragments thereof in cells. Conventional cloning techniques or artificial gene synthesis can be used to generate recombinant expression vectors according to the examples provided herein. Such techniques are well known to those of ordinary skill in the art in view of the present disclosure.

In another general aspect, the invention relates to a host cell comprising a vector comprising a polynucleotide encoding a single-stranded trimeric CD40L Fc fusion protein or a fragment thereof. In another general aspect, the invention relates to a host cell comprising a polynucleotide encoding a single-stranded trimeric CD40L Fc fusion protein or a fragment thereof. Any host cell known to those of ordinary skill in the art in light of this disclosure may be used for recombinant expression of the antibodies or antigen-binding fragments thereof provided herein. In some embodiments, the host cell line is E. coli TG1 or BL21 cells (for expressing, for example, Fc monomeric peptides), CHO-DG44 or CHO-K1 cells, or HEK293 cells (for expressing, for example, single-chain trimeric CD40L Fc fusion protein). According to a specific embodiment, the recombinant expression vector is transformed into a host cell by conventional methods (such as chemical transfection, heat shock, or electroporation), wherein the recombinant expression vector is stably integrated into the genome of the host cell, so that Efficient expression of recombinant nucleic acids.

In another aspect, the present invention provides a pharmaceutical composition comprising the single-chain trimeric CD40L Fc fusion protein described herein, or a pharmaceutically acceptable carrier thereof.

In another aspect, the present invention provides a pharmaceutical composition comprising components for delivering the single-chain trimeric CD40L Fc fusion protein described herein, and a pharmaceutically acceptable carrier.

Also provided is a method of producing a pharmaceutical composition comprising combining a single chain trimeric CD40L Fc fusion protein described herein with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition. The term "pharmaceutical composition" as used herein means a product comprising the single-chain trimeric CD40L Fc fusion protein described herein and a pharmaceutically acceptable carrier. Accordingly, pharmaceutical compositions may comprise the single chain trimeric CD40L Fc fusion proteins provided herein, and compositions comprising them are also useful in the manufacture of medicaments for the therapeutic applications mentioned herein.

The term "carrier" as used herein refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, lipid Body encapsulation, or other materials known in the art for pharmaceutical formulations. It will be appreciated that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the effectiveness of the compositions according to the invention or the biological activity of the compositions provided herein. According to embodiments in view of the present disclosure, any pharmaceutically acceptable carrier suitable for use in a pharmaceutical composition having a fusion protein as an active ingredient can be used herein.

The formulation of pharmaceutically active ingredients and pharmaceutically acceptable carriers is known in the art, eg, Remington: The Science and Practice of Pharmacy (eg, 21st Edition (2005), and any subsequent editions). Non-limiting examples of additional ingredients include: buffers, diluents, solvents, tonicity adjusters, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers can be used to formulate the pharmaceutical compositions provided herein.

In one embodiment of the present invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is an aqueous formulation, ie a formulation comprising water. Liquid formulations may comprise solutions, suspensions, emulsions, microemulsions, gels, and the like. Aqueous formulations generally comprise at least 50% w/w water, or at least 60%, 70%, 75%, 80%, 85%, 90%, or at least 95% w/w water.

In one embodiment, the pharmaceutical composition can be formulated as an injection that can be injected, for example, via an injection device such as a syringe or an infusion pump. For example, injections can be delivered subcutaneously, intramuscularly, intraperitoneally, intravitreally, or intravenously.

In another embodiment, the pharmaceutical composition is a solid formulation, such as a freeze-dried or spray-dried composition, which can be used as it is, or added solvents and/or diluents to it by doctors or patients before use. Solid dosage forms may include tablets such as compressed and/or coated tablets and capsules such as hard or soft gelatin capsules. The pharmaceutical compositions may also be in the form of, for example, sachets, dragees, powders, granules, lozenges, or powders for reconstitution.

The dosage form may be immediate release, in which case it may contain a water-soluble or dispersible carrier, or it may be delayed, sustained, or modified release, in which case it may contain a modulating dosage form in the gastrointestinal tract or in the Water-insoluble polymers that dissolve quickly under the skin.

In other embodiments, the pharmaceutical composition may be delivered intranasally, buccally, or sublingually.

The pH of the aqueous formulation can be between pH 3 and pH 10. In one embodiment provided herein, the pH of the formulation is from about 7.0 to about 9.5. In another embodiment provided herein, the pH of the formulation is from about 3.0 to about 7.0.

In another embodiment provided herein, the pharmaceutical composition comprises a buffer. Non-limiting examples of buffering agents include: arginine, aspartic acid, bicine, citric acid, disodium phosphate, fumaric acid, glycine, glycinyl Glycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, trimethylol glycine (tricine ), and ginseng (hydroxymethyl)-aminomethane, and mixtures thereof. Buffering agents may be present individually or collectively at a concentration of from about 0.01 mg/ml to about 50 mg/ml, such as from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each of these specific buffers constitute alternative embodiments provided herein.

In another embodiment provided herein, the pharmaceutical composition includes a preservative. Non-limiting examples of preservatives include: benzoin chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, Chlorphenacine, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidurea, methyl 4-hydroxybenzoate, phenol, 2-hydroxybenzoate Oxyethanol, 2-phenylethyl alcohol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thiomerosal, and mixtures thereof. Preservatives may be present individually or collectively at a concentration of from about 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each of these specific preservatives constitute alternative embodiments provided herein.

In another embodiment provided herein, the pharmaceutical composition includes an isotonic agent. Non-limiting examples of isotonic agents include salts such as sodium chloride, amino acids such as glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptamine, acid, and threonine), alditols (such as glycerin, 1,2-propanediol (propylene glycol), 1,3-propanediol, and 1,3-butanediol), polyethylene glycols (such as PEG400 ), and mixtures thereof. Another example of isotonic agents includes sugars. Non-limiting examples of sugars may include monosaccharides, disaccharides, or polysaccharides, or water-soluble glucans including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, Pullulan, dextrin, cyclodextrin, alpha and beta-HPCD, soluble starch, hydroxyethyl starch, and sodium carboxymethylcellulose. Another example of an isotonic agent is a sugar alcohol, wherein the term "sugar alcohol" is defined as a C(4-8) hydrocarbon having at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. Isotonic agents may be present individually or collectively at a concentration of from about 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each of these specific isotonic agents constitute alternative embodiments provided herein.

In another embodiment provided herein, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include citric acid, aspartic acid, salts of ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. Chelating agents may be present individually or collectively at a concentration of from about 0.01 mg/ml to about 50 mg/ml, such as from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each of these specific chelating agents constitute alternative embodiments of the invention.

In another embodiment provided herein, the pharmaceutical composition includes a stabilizer. Non-limiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and/or one or more protease inhibitors.

In another embodiment provided herein, the pharmaceutical composition comprises a stabilizer, wherein the stabilizer is carboxy-/hydroxycellulose and its derivatives (such as HPC, HPC-SL, HPC-L and HPMC), cyclodextrin , 2-methylthioethanol, polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (such as sodium chloride), sulfur-containing substances (such as monothioglycerol) , or thioglycolic acid. Stabilizers may be present individually or collectively at a concentration of from about 0.01 mg/ml to about 50 mg/ml, for example from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each of these specific stabilizers constitute alternative embodiments provided herein.

In further embodiments provided herein, the pharmaceutical composition comprises one or more surfactants, preferably one surfactant, at least one surfactant, or two different surfactants. The term "surfactant" refers to any molecule or ion that contains a water-soluble (hydrophilic) portion and a fat-soluble (lipophilic) portion. The surfactant may, for example, be selected from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants, and/or zwitterionic surfactants. Surfactants may be present individually or collectively at a concentration of from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each of these specific surfactants constitute alternative embodiments provided herein.

In further embodiments provided herein, the pharmaceutical composition comprises one or more protease inhibitors, such as, for example, EDTA, and/or benzamidine hydrochloride (HCl). Protease inhibitors may be present individually or collectively at a concentration of from about 0.1 mg/ml to about 20 mg/ml. Pharmaceutical compositions comprising each of these specific protease inhibitors constitute alternative embodiments provided herein.

In another general aspect, the invention relates to a method of producing a pharmaceutical composition comprising a single-chain trimeric CD40L Fc fusion protein disclosed herein or a fragment thereof, the method comprising combining a single-chain trimeric CD40L The Fc fusion protein or fragment is combined with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition. 6. Example

The present invention provides the following non-limiting examples.

In one set of embodiments (Example Group A), there is provided: A1. A single-chain trimeric CD40L Fc fusion protein comprising: (a) three CD40 ligand (CD40L) subunits linked by peptides daughters are covalently linked to each other (CD40L trimer); and (b) Fc monomer peptide. A2. The single-chain trimeric CD40L Fc fusion protein of embodiment A1, wherein the Fc monomer peptide is covalently linked to the CD40L trimer via a peptide tether. A3. The single-chain trimeric CD40L Fc fusion protein of embodiment A2, wherein the peptide tether comprises between 0 and 20 amino acids. A4. The single-chain trimeric CD40L Fc fusion protein of embodiment A1, wherein the CD40 ligand subunits comprise a part of the CD40L extracellular domain. A5. The single-chain trimeric CD40L Fc fusion protein as described in embodiment A1, wherein the CD40L trimer system is linked to the N-terminus of the Fc monomer peptide. A6. The single-chain trimeric CD40L Fc fusion protein of embodiment A5, wherein the single-chain trimeric CD40L Fc fusion protein comprises any sequence selected from SEQ ID NO: 1 to 12, or a fragment thereof. A7. The single-chain trimeric CD40L Fc fusion protein as described in embodiment A1, wherein the CD40L trimer system is linked to the C-terminus of the Fc monomer peptide. A8. The single-chain trimeric CD40L Fc fusion protein of embodiment A7, wherein the single-chain trimeric CD40L Fc fusion protein comprises any sequence selected from SEQ ID NO: 13 to 19, or a fragment thereof. A9. The single-chain trimeric CD40L Fc fusion protein of embodiment A8, wherein the single-chain trimeric CD40L Fc fusion protein comprises SEQ ID NO: 16 or a fragment thereof. A10. The single-chain trimeric CD40L Fc fusion protein of embodiment A1, wherein the CD40 ligand subunits comprise any one of the sequences selected from SEQ ID NO: 20 to 22, or a fragment thereof. A11. The single-chain trimeric CD40L Fc fusion protein of embodiment A1, wherein the Fc monomer peptide comprises a human Fc sequence. A12. The single-chain trimeric CD40L Fc fusion protein of embodiment A11, wherein the human Fc sequence comprises a sequence selected from immunoglobulin IgG, IgA, IgM, IgD, and IgE. A13. The single-chain trimeric CD40L Fc fusion protein of embodiment A12, wherein the human Fc sequence comprises an IgG sequence. A14. The single-chain trimeric CD40L Fc fusion protein of embodiment A13, wherein the IgG sequence is selected from IgG1, IgG2, IgG3, and IgG4. A15. The single-chain trimeric CD40L Fc fusion protein of embodiment A14, wherein the IgG sequence comprises an IgG1 sequence. A16. The single-chain trimeric CD40L Fc fusion protein of embodiment A15, wherein the IgG1 sequence comprises SEQ ID NO: 30 or 31 or a fragment thereof. A17. The single-chain trimeric CD40L Fc fusion protein of embodiment A14, wherein the IgG sequence comprises an IgG2 sequence. A18. The single-chain trimeric CD40L Fc fusion protein of embodiment A17, wherein the IgG2 sequence comprises SEQ ID NO: 29 or a fragment thereof. A19. The single-chain trimeric CD40L Fc fusion protein of embodiment A1, wherein the peptide linker is selected from the group consisting of EGKSSGSGS (SEQ ID NO:23) and (G ₃ S) ₃ (SEQ ID NO:25) . A20. The single-chain trimeric CD40L Fc fusion protein as described in embodiment A2, wherein the peptide tether is selected from (G ₄ S) ₃ (SEQ ID NO: 24), (G ₄ S) ₂ (SEQ ID NO :26), (G ₄ S) ₄ (SEQ ID NO: 27), and the group consisting of G ₄ S (SEQ ID NO: 28). A21. The single-chain trimeric CD40L Fc fusion protein as described in any one of embodiments A1 to A20, wherein the single-chain trimeric CD40L Fc fusion protein enhances activation of the CD40 polypeptide compared to wild-type CD40L. A22. The single-chain trimeric CD40L Fc fusion protein of embodiment A21, wherein the activation of the CD40 polypeptide enhances the immunostimulatory function of dendritic cells, B cells, monocytes, and macrophages. A23. The single-chain trimeric CD40L Fc fusion protein as described in embodiment A22, the activation of the CD40 polypeptide comprises enhanced T cell activation compared to wild-type CD40L. A24. The single-chain trimeric CD40L Fc fusion protein as described in embodiment A23, the activation of the CD40L polypeptide comprises enhanced dendritic cell activation compared to wild-type CD40L. A25. The single-chain trimeric CD40L Fc fusion protein as described in any one of embodiments A1 to A24, wherein the single-chain trimeric CD40L Fc fusion protein enhances anti-tumor activity compared to wild-type CD40L. A26. A dimer comprising two single-chain trimeric CD40L Fc fusion proteins as described in any one of embodiments A1 to A25. A27. The dimer as described in embodiment A26, wherein the dimer system is a homodimer. A28. The dimer as described in embodiment A26, wherein the dimer system is formed by the association of the Fc monomer peptides. A29. A polynucleotide encoding the single-stranded trimeric CD40L Fc fusion protein of any one of embodiments A1 to A25. A30. A vector comprising the polynucleotide of embodiment A29. A31. A host cell comprising the vector of embodiment A30. A32. A host cell comprising the polynucleotide of embodiment A29. A33. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the single-chain trimeric CD40L Fc fusion protein of any one of embodiments A1 to A25. A34. A kit comprising the single-chain trimeric CD40L Fc fusion protein of any one of embodiments A1 to A25. A35. The single chain trimeric CD40L Fc fusion protein of any one of embodiments A1 to A25, or the dimer of any one of embodiments 26 to 28, for use in therapy. A36. The single chain trimeric CD40L Fc fusion protein of any one of embodiments A1 to A25, or the dimer of any one of embodiments 26 to 28, for use in the treatment of a disease or disorder. A37. The single-chain trimeric CD40L Fc fusion protein of any one of embodiments A1 to A25, or the dimer of any one of embodiments 26 to 28, for use in eliminating diseased cells in a subject. A38. The single-chain trimeric CD40L Fc fusion protein as described in embodiment A37, wherein the diseased cell is a cancer cell or a cell infected by a pathogen. A39. The single chain trimeric CD40L Fc fusion protein of any one of embodiments A1 to A25, or the dimer of any one of embodiments 26 to 28, for use in the treatment of cancer. A40. The single-chain trimeric CD40L Fc fusion protein of embodiment A39, wherein the cancer is solid cancer or liquid cancer. A41. The single chain trimeric CD40L Fc fusion protein of embodiment A39, wherein the cancer is selected from the group consisting of melanoma, mesothelioma, advanced solid tumors, and lymphoma. A42. The single chain trimeric CD40L Fc fusion protein of embodiments A1 to A25, or the dimer of any one of embodiments 26 to 28, for use in the treatment of infection. A43. The single-chain trimeric CD40L Fc fusion protein of embodiment A42, wherein the infectious pathogenic system is virus, bacteria, fungus, or parasite. A44. The single-chain trimeric CD40L Fc fusion protein as described in embodiments A1 to A25, or the dimer as described in any one of embodiments 26 to 28, for administration of a vaccine composition. A43. The single-chain trimeric CD40L Fc fusion protein as described in embodiment A44, wherein the vaccine is a vaccine against cancer, infectious pathogens, or allergens.

In another group of embodiments (embodiment group B), there is provided: B1. A system comprising components for providing a single-chain trimeric CD40L Fc fusion protein, the single-chain trimeric CD40L Fc fusion protein comprising: (a ) three CD40L subunits covalently linked to each other by a peptide linker (CD40L trimer); and (b) Fc monomeric peptide. B2. The system of embodiment B1, wherein the Fc monomer peptide is covalently linked to the CD40L trimer by a peptide tether. B3. The system of embodiment B2, wherein the peptide tether comprises between 0 and 20 amino acids. B4. The system of embodiment B1, wherein the CD40 ligand subunits comprise a portion of the CD40L extracellular domain. B5. The system as described in embodiment B1, wherein the CD40L trimer system is linked to the N-terminus of the Fc monomer peptide. B6. The system according to embodiment B5, wherein the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 1 to 12, or a fragment thereof. B7. The system as described in embodiment B1, wherein the CD40L trimer system is linked to the C-terminus of the Fc monomer peptide. B8. The system according to embodiment B7, wherein the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 13 to 19, or a fragment thereof. B9. The system of embodiment B8, wherein the single-chain trimeric CD40L Fc fusion protein comprises SEQ ID NO: 16 or a fragment thereof. B10. The system of embodiment B1, wherein the CD40 ligand subunits comprise any one of the sequences selected from SEQ ID NOS: 20 to 22, or a fragment thereof. B11. The system of embodiment B1, wherein the Fc monomer peptide comprises a human Fc sequence. B12. The system of embodiment B11, wherein the human Fc sequence comprises a sequence selected from immunoglobulin IgG, IgA, IgM, IgD, and IgE. B13. The system of embodiment B12, wherein the human Fc sequence comprises an IgG sequence. B14. The system of embodiment B13, wherein the IgG sequence is selected from IgG1, IgG2, IgG3, and IgG4. B15. The system of embodiment B14, wherein the IgG sequence is an IgG1 sequence. B16. The system of embodiment B15, wherein the IgG1 sequence comprises SEQ ID NO: 30 or 31 or a fragment thereof. B17. The system of embodiment B14, wherein the IgG sequence comprises an IgG2 sequence. B18. The system of embodiment B17, wherein the IgG2 sequence comprises SEQ ID NO: 29 or a fragment thereof. B19. The system of embodiment B1, wherein the peptide linker is selected from the group consisting of EGKSSGSGS (SEQ ID NO: 23) and (G ₃ S) ₃ (SEQ ID NO: 25). B20. The system of embodiment B2, wherein the peptide tether is selected from (G ₄ S) ₃ (SEQ ID NO:24), (G ₄ S) ₂ (SEQ ID NO:26), (G ₄ The group consisting of S) ₄ (SEQ ID NO:27), and G ₄ S (SEQ ID NO:28). B21. The system of any one of embodiments B1 to B20, wherein the single-chain trimeric CD40L Fc fusion protein enhances activation of the CD40 polypeptide compared to wild-type CD40L. B22. The system of embodiment B21, wherein the activation of the CD40 polypeptide enhances the immunostimulatory function of dendritic cells, B cells, monocytes, and macrophages. B23. The system of embodiment B22, wherein the activation of the CD40 polypeptide comprises enhanced T cell activation compared to wild-type CD40L. B24. The system of embodiment B22, wherein the activation of the CD40 polypeptide comprises enhanced dendritic cell activation compared to wild-type CD40L. B25. The system of any one of embodiments B1 to B24, wherein the single-chain trimeric CD40L Fc fusion protein comprises enhanced anti-tumor activity compared to wild-type CD40L. B26. A system comprising a building block for providing a dimer comprising two single-chain trimeric CD40L Fc fusion proteins each comprising: (a) three CD40L subunits formed by Peptide linkers are covalently linked to each other; and (b) Fc monomer peptides. B27. The system of embodiment B26, wherein the dimer system is a homodimer. B28. The system of embodiment B26, wherein the dimerization system is formed by association of the Fc monomeric peptides. B29. A system comprising means for providing a polynucleotide encoding the single-stranded trimeric CD40L Fc fusion protein of any one of embodiments B1 to B25. B30. A system comprising means for providing a vector comprising the polynucleotide of embodiment B29. B31. A system comprising means for providing a host cell comprising the vector of embodiment B30. B32. A system comprising means for providing a host cell comprising the polynucleotide of embodiment B29. B33. A system comprising means for providing a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the single-chain trimeric CD40L Fc fusion protein of any one of embodiments B1 to B25. B34. A system comprising means for providing a kit comprising the single-chain trimeric CD40L Fc fusion protein of any one of embodiments B1 to B25.

In another group of embodiments (embodiment group C), it provides: C1. A method for activating a CD40 polypeptide, comprising contacting the CD40 polypeptide with a single-chain trimeric CD40L Fc fusion protein, the single-chain trimeric CD40L Fc fusion The protein comprises: (a) three CD40L subunits covalently linked to each other by a peptide linker (CD40L trimer); and (b) an Fc monomeric peptide, wherein the single-chain trimeric CD40L Fc fusion protein is in Activates CD40 polypeptide upon binding. C2. The method of embodiment C1, wherein the Fc monomer peptide is covalently linked to the CD40L trimer by a peptide tether. C3. The method of embodiment C2, wherein the peptide tether comprises between 0 and 20 amino acids. C4. The method of embodiment C1, wherein the CD40 ligand subunits comprise a portion of the CD40L extracellular domain. C5. The method as described in embodiment C1, wherein the CD40L trimer system is linked to the N-terminus of the Fc monomer peptide. C6. The method as described in embodiment C5, wherein the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 1 to 12, or a fragment thereof. C7. The method as described in embodiment C1, wherein the CD40L trimer system is linked to the C-terminus of the Fc monomer peptide. C8. The method according to embodiment C7, wherein the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 13 to 19, or a fragment thereof. C9. The method of embodiment C8, wherein the single-chain trimeric CD40L Fc fusion protein comprises SEQ ID NO: 16 or a fragment thereof. C10. The method of embodiment C1, wherein the CD40 ligand subunits comprise any one of the sequences selected from SEQ ID NOS: 20 to 22, or a fragment thereof. C11. The method of embodiment C1, wherein the Fc monomer peptide comprises a human Fc sequence. C12. The method of embodiment C11, wherein the human Fc sequence comprises a sequence selected from immunoglobulin IgG, IgA, IgM, IgD, and IgE. C13. The method of embodiment C12, wherein the human Fc sequence comprises an IgG sequence. C14. The method of embodiment C13, wherein the IgG sequence is selected from IgG1, IgG2, IgG3, and IgG4. C15. The method of embodiment C14, wherein the IgG sequence is an IgG1 sequence. C16. The method of embodiment C15, wherein the IgG1 sequence comprises SEQ ID NO: 30 or 31 or a fragment thereof. C17. The method of embodiment C14, wherein the IgG sequence is an IgG2 sequence. C18. The method of embodiment C17, wherein the IgG2 sequence comprises SEQ ID NO: 29 or a fragment thereof. C19. The method of embodiment C1, wherein the peptide linker is selected from the group consisting of EGKSSGSGS (SEQ ID NO: 23) and (G ₃ S) ₃ (SEQ ID NO: 25). C20. The method of embodiment C2, wherein the peptide tether is selected from (G ₄ S) ₃ (SEQ ID NO:24), (G ₄ S) ₂ (SEQ ID NO:26), (G ₄ The group consisting of S) ₄ (SEQ ID NO:27), and G ₄ S (SEQ ID NO:28). C21. The method of any one of embodiments C1 to C20, comprising enhanced activation of the CD40 polypeptide compared to wild-type CD40L. C22. The method of embodiment C21, wherein the activation of the CD40 polypeptide enhances the immunostimulatory function of dendritic cells, B cells, monocytes, and macrophages. C23. The method of embodiment C21, wherein the activation of the CD40 polypeptide comprises enhanced T cell activation compared to wild-type CD40L. C24. The method of embodiment C21, wherein the activation of the CD40 polypeptide comprises enhanced dendritic cell activation compared to wild-type CD40L. C25. The method of any one of embodiments C1 to C24, comprising enhanced anti-tumor activation compared to wild-type CD40L. C26. A method of activating a CD40 polypeptide comprising contacting the CD40 polypeptide with a dimer comprising two single-chain trimeric CD40L Fc fusion proteins each comprising: (a) three CD40L subunits, which are covalently linked to each other by a peptide linker; and (b) an Fc monomeric peptide, wherein the single chain trimeric CD40L Fc fusion protein dimer activates the CD40 polypeptide upon binding. C27. The method of embodiment C26, further comprising administering a homodimer. C28. The method of embodiment C26, wherein the dimerization system is formed by association of the Fc monomeric peptides. C29. The method of any one of embodiments C1 to C28, wherein the method is performed in vitro. C30. The method of any one of embodiments C1 to C28, wherein the method is performed in vivo. C31. The method of embodiment C30, further wherein the contacting comprises administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the single-chain trimeric CD40L Fc fusion protein. C32. The method of embodiment C31, wherein the contacting enhances an innate anti-tumor immune response. C33. A method of activating T cells, comprising contacting the T cells with antigen-presenting cells in the presence of a single-chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits units, which are covalently linked to each other by a peptide linker (CD40L trimer); and (b) Fc monomeric peptide, wherein the antigen presenting cell expresses the CD40 polypeptide; and wherein the single chain trimeric CD40L Fc fusion protein is in Activates T cells upon binding to CD40 polypeptide. C34. The method of embodiment C33, wherein the Fc monomer peptide is covalently linked to the CD40L trimer by a peptide tether. C35. The method of embodiment C34, wherein the peptide tether comprises between 0 and 20 amino acids. C36. The method of embodiment C33, wherein the CD40 ligand subunits comprise a portion of the CD40L extracellular domain. C37. The method according to embodiment C33, wherein the CD40L trimer is linked to the N-terminus of the Fc monomer peptide. C38. The method according to embodiment C37, wherein the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 1 to 12, or a fragment thereof. C39. The method as described in embodiment C33, wherein the CD40L trimer system is linked to the C-terminus of the Fc monomer peptide. C40. The method according to embodiment C39, wherein the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 13 to 19, or a fragment thereof. C41. The method of embodiment C40, wherein the single-chain trimeric CD40L Fc fusion protein comprises SEQ ID NO: 16 or a fragment thereof. C42. The method of embodiment C33, wherein the CD40 ligand subunits comprise any one of the sequences selected from SEQ ID NOS: 20 to 22, or a fragment thereof. C43. The method of embodiment C33, wherein the Fc monomer peptide comprises a human Fc sequence. C44. The method of embodiment C43, wherein the human Fc sequence comprises a sequence selected from immunoglobulin IgG, IgA, IgM, IgD, and IgE. C45. The method of embodiment C44, wherein the human Fc sequence comprises an IgG sequence. C46. The method of embodiment C45, wherein the IgG sequence is selected from IgG1, IgG2, IgG3, and IgG4. C47. The method of embodiment C46, wherein the IgG sequence is an IgG1 sequence. C48. The method of embodiment C47, wherein the IgG1 sequence comprises SEQ ID NO: 30 or 31 or a fragment thereof. C49. The method of embodiment C46, wherein the IgG sequence is an IgG2 sequence. C50. The method of embodiment C49, wherein the IgG2 sequence comprises SEQ ID NO: 29 or a fragment thereof. C51. The method of embodiment C33, wherein the peptide linker is selected from the group consisting of EGKSSGSGS (SEQ ID NO:23) and (G3S)3 (SEQ ID NO:25). C52. The method of embodiment C34, wherein the peptide tether is selected from (G ₄ S) ₃ (SEQ ID NO:24), (G ₄ S) ₂ (SEQ ID NO:26), (G ₄ The group consisting of S) ₄ (SEQ ID NO:27), and G ₄ S (SEQ ID NO:28). C53. The method of any one of embodiments C33 to C52, further comprising enhanced activation of the CD40 polypeptide compared to wild-type CD40L. C54. The method of embodiment C53, wherein the enhanced activation of the CD40 polypeptide enhances the immunostimulatory function of dendritic cells, B cells, monocytes, and macrophages. C55. The method of embodiment C53, further comprising enhancing T cell activation compared to wild-type CD40L. C56. The method of embodiment C53, further comprising enhancing dendritic cell activation compared to wild-type CD40L. C57. The method of any one of embodiments C33 to C56, wherein the single-chain trimeric CD40L Fc fusion protein comprises enhanced anti-tumor activity compared to wild-type CD40L. C58. A method of activating T cells, comprising contacting the CD40 polypeptide with an antigen-presenting cell in the presence of a dimer comprising two single-chain trimeric CD40L Fc fusion proteins each comprising: (a) Three CD40L subunits, which are covalently linked to each other by peptide linkers; and (b) Fc monomeric peptides, wherein the antigen presenting cells express the CD40 polypeptide, wherein the single-chain trimeric CD40L Fc fusion protein dimer binds CD40 peptide activates T cells. C59. The method of embodiment C58, further comprising administering a homodimer. C60. The method of embodiment C58, wherein the dimerization system is formed by association of the Fc monomer peptides. C61. The method of any one of embodiments C33 to C60, wherein the method is performed in vitro. C62. The method of any one of embodiments C33 to C60, wherein the method is performed in vivo. C63. The method of any one of embodiments C62, further wherein the contacting comprises administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the single-chain trimeric CD40L Fc fusion protein. C64. The method of embodiment C63, wherein the contacting enhances an innate anti-tumor immune response. C65. A method of activating dendritic cells, comprising contacting the CD40 polypeptide with a single-chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits by covalently linked to each other by a peptide linker (CD40L trimer); and (b) an Fc monomeric peptide, wherein the single chain trimeric CD40L Fc fusion protein activates dendritic cells upon binding the CD40 polypeptide. C66. The method of embodiment C65, wherein the Fc monomer peptide is covalently linked to the CD40L trimer by a peptide tether. C67. The method of embodiment C66, wherein the peptide tether comprises between 0 and 20 amino acids. C68. The method of embodiment C65, wherein the CD40 ligand subunits comprise a portion of the CD40L extracellular domain. C69. The method of embodiment C65, wherein the CD40L trimer is linked to the N-terminus of the Fc monomer peptide. C70. The method according to embodiment C69, wherein the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 1 to 12, or a fragment thereof. C71. The method as described in embodiment C65, wherein the CD40L trimer system is linked to the C-terminus of the Fc monomer peptide. C72. The method according to embodiment C71, wherein the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 13 to 19, or a fragment thereof. C73. The method of embodiment C72, wherein the single-chain trimeric CD40L Fc fusion protein comprises SEQ ID NO: 16 or a fragment thereof. C74. The method of embodiment C65, wherein the CD40 ligand subunits comprise any one of the sequences selected from SEQ ID NOS: 20 to 22, or a fragment thereof. C75. The method of embodiment C65, wherein the Fc monomer peptide comprises a human Fc sequence. C76. The method of embodiment C75, wherein the human Fc sequence comprises a sequence selected from immunoglobulin IgG, IgA, IgM, IgD, and IgE. C77. The method of embodiment C76, wherein the human Fc sequence comprises an IgG sequence. C78. The method of embodiment C77, wherein the IgG sequence is selected from IgG1, IgG2, IgG3, and IgG4. C79. The method of embodiment C78, wherein the IgG sequence is an IgG1 sequence. C80. The method of embodiment C79, wherein the IgG1 sequence comprises SEQ ID NO: 30 or 31 or a fragment thereof. C81. The method of embodiment C78, wherein the IgG sequence is an IgG2 sequence. C82. The method of embodiment C81, wherein the IgG2 sequence comprises SEQ ID NO: 29 or a fragment thereof. C83. The method of embodiment C65, wherein the peptide linker is selected from the group consisting of EGKSSGSGS (SEQ ID NO: 23) and (G ₃ S) ₃ (SEQ ID NO: 25). C84. The method of embodiment C66, wherein the peptide tether is selected from the group consisting of (G ₄ S) ₃ (SEQ ID NO:24), (G ₄ S) ₂ (SEQ ID NO:26), (G ₄ The group consisting of S) ₄ (SEQ ID NO:27), and G ₄ S (SEQ ID NO:28). C85. The method of any one of embodiments C65 to C84, comprising enhanced activation of the CD40 polypeptide compared to wild-type CD40L. C86. The method of embodiment C85, wherein the enhanced activation of the CD40 polypeptide enhances the immunostimulatory function of dendritic cells, B cells, monocytes, and macrophages. C87. The method of embodiment C85, further comprising enhancing T cell activation compared to wild-type CD40L. C88. The method of embodiment C85, further comprising enhancing dendritic cell activation compared to wild-type CD40L. C89. The method of any one of embodiments C65 to C88, wherein the single-chain trimeric CD40L Fc fusion protein comprises enhanced anti-tumor activity compared to wild-type CD40L. C90. A method of activating dendritic cells comprising contacting the CD40 polypeptide with a dimer comprising two single-chain trimeric CD40L Fc fusion proteins each comprising: (a) three CD40L subunits , which are covalently linked to each other by a peptide linker; and (b) an Fc monomeric peptide, wherein the single-chain trimeric CD40L Fc fusion protein activates dendritic cells when bound to a CD40 polypeptide. C91. The method of embodiment C90, further comprising administering a homodimer. C92. The method of embodiment C90, wherein the dimerization system is formed by association of the Fc monomer peptides. C93. The method of any one of embodiments C54 to C92, wherein the method is performed in vitro. C94. The method of any one of embodiments C54 to C92, wherein the method is performed in vivo. C95. The method of any one of embodiments C94, further wherein the contacting comprises administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the single-chain trimeric CD40L Fc fusion protein. C96. The method of embodiment C95, wherein the contacting enhances an innate anti-tumor immune response.

In another group of embodiments (embodiment group D), provide: D1. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of single-chain trimeric CD40L Fc fusion protein, comprising: ( a) Three CD40L subunits covalently linked to each other by a peptide linker (CD40L trimer); and (b) Fc monomeric peptide. D2. The method of embodiment D1, wherein the Fc monomer peptide is covalently linked to the CD40L trimer by a peptide tether. D3. The method of embodiment D2, wherein the peptide tether comprises between 0 and 20 amino acids. D4. The method of embodiment D1, wherein the CD40 ligand subunits comprise a portion of the CD40L extracellular domain. D5. The method as described in embodiment D1, wherein the CD40L trimer system is linked to the N-terminus of the Fc monomer peptide. D6. The method as described in embodiment D5, wherein the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 1 to 12, or a fragment thereof. D7. The method as described in embodiment D1, wherein the CD40L trimer system is linked to the C-terminus of the Fc monomer peptide. D8. The method as described in embodiment D7, wherein the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 13 to 19, or a fragment thereof. D9. The method according to embodiment D8, wherein the single-chain trimeric CD40L Fc fusion protein comprises SEQ ID NO: 16 or a fragment thereof. D10. The method of embodiment D1, wherein the CD40 ligand subunits comprise any one of the sequences selected from SEQ ID NOS: 20 to 22, or a fragment thereof. D11. The method of embodiment D1, wherein the Fc monomer peptide comprises a human Fc sequence. D12. The method of embodiment D11, wherein the human Fc sequence comprises a sequence selected from immunoglobulin IgG, IgA, IgM, IgD, and IgE. D13. The method of embodiment D12, wherein the human Fc sequence comprises an IgG sequence. D14. The method of embodiment D13, wherein the IgG sequence is selected from IgG1, IgG2, IgG3, and IgG4. D15. The method of embodiment D14, wherein the IgG sequence is an IgG1 sequence. D16. The method according to embodiment D15, wherein the IgG1 sequence comprises SEQ ID NO: 30 or 31 or a fragment thereof. D17. The method of embodiment D14, wherein the IgG sequence is an IgG2 sequence. D18. The method of embodiment D17, wherein the IgG2 sequence comprises SEQ ID NO: 29 or a fragment thereof. D19. The method of embodiment D1, wherein the peptide linker is selected from the group consisting of EGKSSGSGS (SEQ ID NO: 23) and (G ₃ S) ₃ (SEQ ID NO: 25). D20. The method of embodiment D2, wherein the peptide tether is selected from (G ₄ S) ₃ (SEQ ID NO:24), (G ₄ S) ₂ (SEQ ID NO:26), (G ₄ The group consisting of S) ₄ (SEQ ID NO:27), and G ₄ S (SEQ ID NO:28). D21. The method of any one of embodiments D1 to D20, wherein the single-chain trimeric CD40L Fc fusion protein comprises enhanced activation of the CD40 polypeptide compared to wild-type CD40L. D22. The method according to embodiment D21, wherein the enhanced activation of the CD40 polypeptide enhances the immunostimulatory function of dendritic cells, B cells, monocytes, and macrophages. D23. The method of embodiment D22, wherein the enhanced activation of the CD40 polypeptide comprises enhanced T cell activation compared to wild-type CD40L. D24. The method of embodiment D22, wherein the single-chain trimeric CD40L Fc fusion protein comprises enhanced dendritic cell activation compared to wild-type CD40L. D25. The method of any one of embodiments D1 to D24, wherein the single-chain trimeric CD40L Fc fusion protein comprises enhanced anti-tumor activity compared to wild-type CD40L. D26. A method of treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a dimer comprising two single-chain trimeric CD40L Fc fusion proteins, each comprising: (a) three CD40L subunits, which are covalently linked to each other by a peptide linker; and (b) an Fc monomer peptide. D27. The method of embodiment D26, further comprising administering a homodimer. D28. The method of embodiment D26, wherein the dimerization system is formed by the association of the Fc monomer peptides. D29. The method of any one of embodiments D1 to D28, further comprising administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the single-chain trimeric CD40L Fc fusion protein. D30. The method of any one of embodiments D1 to D29, wherein the treatment enhances an innate anti-tumor immune response. D31. The method of any one of embodiments D1 to D30, further comprising co-administration of a second therapy. D32. The method of any one of embodiments D1 to D31, wherein the cancer is selected from the group consisting of melanoma, mesothelioma, advanced solid tumors, and lymphoma.

In another group of embodiments (Embodiment Group E), there is provided: E1. A method for producing a single-stranded trimeric CD40L Fc fusion protein or a fragment thereof, comprising: (a) introducing a polynucleotide into a host cell, The polynucleotide encodes a single-chain trimeric CD40L Fc fusion protein comprising (i) three CD40L subunits covalently linked to each other by a peptide linker (CD40L trimer); and (ii) Fc monomeric peptide; (b) culturing the host cell under conditions to produce the single-chain trimeric CD40L Fc fusion protein or fragment thereof; and (c) from the cell or culture The single-chain trimeric CD40L Fc fusion protein or a fragment thereof is recovered. E2. The method of embodiment E1, wherein the Fc monomer peptide is covalently linked to the CD40L trimer by a peptide tether. E3. The method of embodiment E2, wherein the peptide tether comprises between 0 and 20 amino acids. E4. The method of embodiment E1, wherein the CD40 ligand subunits comprise a portion of the CD40L extracellular domain. E5. The method as described in embodiment E1, wherein the CD40L trimer system is linked to the N-terminus of the Fc monomer peptide. E6. The method according to embodiment E5, wherein the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 1 to 12, or a fragment thereof. E7. The method as described in embodiment E1, wherein the CD40L trimer system is linked to the C-terminus of the Fc monomer peptide. E8. The method according to embodiment E7, wherein the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 13 to 19, or a fragment thereof. E9. The method of embodiment E8, wherein the single-chain trimeric CD40L Fc fusion protein comprises SEQ ID NO: 16 or a fragment thereof. E10. The method of embodiment E1, wherein the CD40 ligand subunits comprise any one of the sequences selected from SEQ ID NOS: 20 to 22, or a fragment thereof. E11. The method of embodiment E1, wherein the Fc monomer peptide comprises a human Fc sequence. E12. The method of embodiment E11, wherein the human Fc sequence comprises a sequence selected from immunoglobulin IgG, IgA, IgM, IgD, and IgE. E13. The method of embodiment E12, wherein the human Fc sequence comprises an IgG sequence. E14. The method of embodiment E13, wherein the IgG sequence is selected from IgG1, IgG2, IgG3, and IgG4. E15. The method of embodiment E14, wherein the IgG sequence is an IgG1 sequence. E16. The method of embodiment E15, wherein the IgG1 sequence comprises SEQ ID NO: 30 or 31 or a fragment thereof. E17. The method of embodiment E14, wherein the IgG sequence is an IgG2 sequence. E18. The method of embodiment E17, wherein the IgG2 sequence comprises SEQ ID NO: 29 or a fragment thereof. E19. The method of embodiment E1, wherein the peptide linker is selected from the group consisting of EGKSSGSGS (SEQ ID NO: 23) and (G ₃ S) ₃ (SEQ ID NO: 25). E20. The method of embodiment E2, wherein the peptide tether is selected from (G ₄ S) ₃ (SEQ ID NO:24), (G ₄ S) ₂ (SEQ ID NO:26), (G ₄ The group consisting of S) ₄ (SEQ ID NO:27), and G ₄ S (SEQ ID NO:28). E21. The method of any one of embodiments E1 to E20, wherein the single-chain trimeric CD40L Fc fusion protein enhances activation of the CD40 polypeptide compared to wild-type CD40L. E22. The method of embodiment E21, wherein the activation of the CD40 polypeptide enhances the immunostimulatory function of dendritic cells, B cells, monocytes, and macrophages. E23. The method of embodiment E22, wherein the activation of the CD40 polypeptide comprises enhanced T cell activation compared to wild-type CD40L. E24. The method of embodiment E23, wherein the activation of the CD40 polypeptide comprises enhanced dendritic cell activation compared to wild-type CD40L. E25. The method of embodiment E1, wherein the single-chain trimeric CD40L Fc fusion protein comprises enhanced anti-tumor activity compared to wild-type CD40L. E26. Provided herein is a method of producing a dimer comprising two single-stranded trimeric CD40L Fc fusion proteins, each method comprising: (a) introducing into a host cell a polynucleotide encoding a single A chain trimeric CD40L Fc fusion protein, the single chain trimeric CD40L Fc fusion protein comprising (i) three CD40L subunits covalently linked to each other by a peptide linker; and (ii) an Fc monomer (b) cultivating the host cell under conditions to produce the single-chain trimeric CD40L Fc fusion protein or fragment thereof; (c) recovering the single-chain trimeric CD40L Fc fusion protein or fragment thereof from the cell or culture and (d) combining a single-chain trimeric CD40L Fc fusion protein or fragment thereof under conditions favorable for dimerization. E27. The method of embodiment E26, wherein the dimer comprises a homodimer. E28. The method of embodiment E26, wherein the dimerization system is formed by association of the Fc monomeric peptides. E29. A method of producing a pharmaceutical composition having a single-chain trimeric CD40L Fc fusion protein or fragment thereof, comprising combining the single-chain trimeric CD40L Fc fusion protein or fragment thereof produced according to any one of embodiments E1 to E25 with A pharmaceutically acceptable carrier is combined to obtain the pharmaceutical composition.

In another group of embodiments (embodiment group F), it provides: F1. A method of increasing an immune response in a subject, comprising administering to the subject a therapeutically effective amount of a single-chain trimeric CD40L Fc fusion protein, comprising: (a) Three CD40L subunits covalently linked to each other by a peptide linker; and (b) Fc monomer peptide. F2. A method of increasing an immune response to a cancer antigen in a subject having cancer, comprising administering to the subject a therapeutically effective amount of a single-chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits units, which are covalently linked to each other by a peptide linker; and (b) an Fc monomer peptide. F3. A method of increasing an immune response to a pathogen in a subject infected with the pathogen, comprising administering to the subject a therapeutically effective amount of a single-chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits units, which are covalently linked to each other by a peptide linker; and (b) an Fc monomer peptide. F4. A method of increasing an immune response to a vaccine antigen in a subject administered the vaccine, wherein the method further comprises administering to the subject a therapeutically effective amount of a single-chain trimeric CD40L Fc fusion protein comprising: (a) Three CD40L subunits covalently linked to each other by a peptide linker; and (b) Fc monomer peptide. F5. The method of any one of embodiments F1 to F4, wherein the peptide tether comprises between 0 and 20 amino acids. F6. The method of any one of embodiments F1 to F5, wherein the CD40 ligand subunits comprise a portion of the CD40L extracellular domain. F7. The method of any one of embodiments F1 to F6, wherein the CD40L trimer is linked to the N-terminus of the Fc monomeric peptide. F8. The method according to any one of embodiments F1 to F7, wherein the single-chain trimeric CD40L Fc fusion protein comprises any sequence selected from SEQ ID NO: 1 to 12, or a fragment thereof. F9. The method of any one of embodiments F1 to F8, wherein the CD40L trimer is linked to the C-terminus of the Fc monomeric peptide. F10. The method according to any one of embodiments F1 to F9, wherein the single-chain trimeric CD40L Fc fusion protein comprises any sequence selected from SEQ ID NO: 13 to 19, or a fragment thereof. F11. The method of any one of embodiments F1 to F10, wherein the single-chain trimeric CD40L Fc fusion protein comprises SEQ ID NO: 16 or a fragment thereof. F12. The method of any one of embodiments F1 to F11, wherein the CD40 ligand subunits comprise any one of the sequences selected from SEQ ID NOS: 20 to 22, or a fragment thereof. F13. The method of any one of embodiments F1 to F12, wherein the Fc monomeric peptide comprises a human Fc sequence. F14. The method of embodiment F13, wherein the human Fc sequence comprises a sequence selected from immunoglobulin IgG, IgA, IgM, IgD, and IgE. F15. The method of embodiment F14, wherein the human Fc sequence comprises an IgG sequence. F16. The method of embodiment F15, wherein the IgG sequence is selected from IgG1, IgG2, IgG3, and IgG4. F17. The method of embodiment F16, wherein the IgG sequence is an IgG1 sequence. F18. The method of embodiment F17, wherein the IgG1 sequence comprises SEQ ID NO: 30 or 31 or a fragment thereof. F19. The method of embodiment F16, wherein the IgG sequence is an IgG2 sequence. F20. The method of embodiment F19, wherein the IgG2 sequence comprises SEQ ID NO: 29 or a fragment thereof. F21. The method as described in any one of embodiments F1 to F20, wherein the peptide linker is selected from the group consisting of EGKSSGSGS (SEQ ID NO:23) and (G ₃ S) ₃ (SEQ ID NO:25) group. F22. The method of any one of embodiments F1 to F20, wherein the peptide tether is selected from (G ₄ S) ₃ (SEQ ID NO: 24), (G ₄ S) ₂ (SEQ ID NO: 26), the group consisting of (G ₄ S) ₄ (SEQ ID NO: 27), and G ₄ S (SEQ ID NO: 28). F23. The method of any one of embodiments F1 to F22, wherein the single-chain trimeric CD40L enhances activation. F24. The method of embodiment F23, wherein the enhanced activation of the CD40 polypeptide enhances the immunostimulatory function of dendritic cells, B cells, monocytes, and macrophages. F25. The method of embodiment F24, wherein the enhanced activation of the CD40 polypeptide comprises enhanced T cell activation compared to wild-type CD40L. F26. The method of embodiment F22, wherein the single-chain trimeric CD40L Fc fusion protein comprises enhanced dendritic cell activation compared to wild-type CD40L. F27. The method of any one of embodiments F1 to F26, wherein the single-chain trimeric CD40L Fc fusion protein comprises enhanced anti-tumor activity compared to wild-type CD40L. F28. A method of increasing an immune response in a subject, comprising administering to the subject a therapeutically effective amount of a dimer comprising two single-chain trimeric CD40L Fc fusion proteins, each comprising: (a) three a CD40L subunit whose members are covalently linked to each other by a peptide linker; and (b) an Fc monomer peptide. F29. A method of increasing an immune response to a cancer antigen in a subject having cancer comprising administering to the subject a therapeutically effective amount of a dimer comprising two single-chain trimeric CD40L Fc fusion proteins , each comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and (b) an Fc monomeric peptide. F30. A method of increasing an immune response to a pathogen in a subject infected with the pathogen comprising administering to the subject a therapeutically effective amount of a dimer comprising two single-chain trimeric CD40L Fc fusion proteins , each comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and (b) an Fc monomeric peptide. F31. A method of increasing an immune response to a vaccine antigen in a subject administered the vaccine, wherein the method further comprises administering to the subject a therapeutically effective amount of a dimer comprising two single-chain trimers CD40L Fc fusion proteins, each comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker; and (b) an Fc monomeric peptide. F32. The method of any one of embodiments F28 to F31, further comprising administering a homodimer. F33. The method of any one of embodiments F28 to F32, wherein the dimerization system is formed by association of the Fc monomeric peptides. F34. The method of any one of embodiments F28 to F33, further comprising administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the single-chain trimeric CD40L Fc fusion protein. F35. The method of any one of embodiments F28-F34, further comprising co-administration of a second therapy.

Specific embodiments of the invention are described herein. Variations of the disclosed embodiments may become apparent to, and are expected to employ, as appropriate, those of ordinary skill in the art upon reading the foregoing description. Accordingly, the invention is intended to be practiced otherwise than as specifically described herein and is intended to include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Several embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the descriptions in the Examples section are intended to illustrate but not limit the scope of the invention described in the Claims. 7. Examples Example 1: Methods and Materials

Design of soluble trimeric CD40L Fc fusion constructs: CD40L is a membrane protein consisting of an extracellular domain, a stalk region, a transmembrane helix, and a short cytoplasmic domain. The activity of CD40L is located in the extracellular domain, and this domain was selected for the generation of Fc fusion constructs. CD40L ECD (Uniprot ID: P29965) was fused with a flexible linker and tether to form N-terminal fusions with effector function silent human IgG1 and human IgG2 isotypes. The linker between CD40L subunits was 9 or 12 amino acids, which was selected for flexibility (although other lengths were not tested). The tether between the last CD40L subunit and Fc was chosen to promote flexibility and ranged from 0 to 20 amino acids. In addition, a C-terminal Fc fusion of CD40L was generated. A flexible tether ranging from 5 to 20 amino acids was placed between the C-terminus of the Fc and the N-terminus of the first CD40L subunit. Between CD40L subunits, flexible linkers of 9 or 12 amino acids were used. Construction of C-terminal trimeric CD40L Fc fusions on effector silenced human IgG1 and IgG2A isotypes. Initial attempts to design the agonist CD40L with CD40L trimer bundles fused to huIgG molecules yielded high molecular weight species (HMWS) after protein A purification, which were not suitable for further development. Using structure-based principles to redesign proteins to optimize their biophysical and functional properties. Proteins were designed with various intra-subunit linkers, tethers, and Fc attachment points ( FIG. 1A ).

In addition, to test the hypothesis that the CD40L trimeric fusion protein is itself active (i.e., forms a dimer with two copies of the CD40L trimeric fusion protein without interaction between its Fc parts), a and construct molecules containing a CD40L trimer fused to an Fc monomeric peptide with mutations in the CH3 domain that prevent dimer formation (“single Fc” molecules, please See eg Figure 1B ). Example 2: Expression and purification

Expi293F cells were grown in serum-free Expi293™ Expression Medium (Invitrogen). Cells were maintained in Erlenmeyer flasks at 37°C with 8% CO2 on an orbital shaker. Cells were transfected at 3.0 x ¹⁰⁶ cells/ml. Plastid DNA was diluted in Opti-MEM I. Dilute Expifectamine 293 in Opti-MEM I and incubate at room temperature for 5 min. The DNA mixture was incubated with Expifectamine 293 and incubated at room temperature for 20 to 30 min. Add the DNA/Expifectamine 293 complex to the cell culture and return to the incubator. 16 to 18 h after transfection, an appropriate volume of the enhancer is added to the cell culture. Cell culture supernatants were harvested on day 6 by centrifugation at 3000g for 10 minutes to pellet the cells. Supernatants were stored at 4°C until purification.

Protein was purified using RoboColumn Eshmuno A, 0.6 ml (MERCK MILLIPORE, Cat.No.1.25163.0001) column and Tecan liquid handler. The supernatant was applied to the column at a flow rate of 0.6 mL/min for maximum capture. Wash the column with 8 column volumes of PBS until a clean baseline (as monitored by UV A280) is obtained. The protein was eluted from the column with 50 mM citrate (pH 3.0), and the protein was neutralized with 1M Tris-HCl (pH 9.0). Desalt the protein in dPBS as the final buffer using a Zebra spin desalting column. Using native and denaturing SDS PAGE gels, test fractions for the presence of recombinant protein and pool the fractions. According to SDS PAGE and analytical size exchange column (aSEC), the purity of the purified protein was considered to be >80%. The second step of chromatographic purification was achieved by Superdex200 gel filtration chromatography using PBS mobile phase. Fractions corresponding to ~150 kDa were pooled and stored at -80 °C until use.

The newly designed trimers showed a significant improvement in purity as judged by monodisperse Fc homodimer levels using HPLC (aSEC) ( Table 1 below) and SDS-PAGE gels ( Figure 2A -2B ). [Table 1.] Monodispersity of engineered CD40L trimers fused to Fc measured by aSEC after protein A purification protein AA Monomer % after protein A purification TPP000182980 79.36 TPP000182981 79.01 TPP000182982 73.61 TPP000182983 81.2 TPP000182984 77.2 TPP000182985 77.68 TPP000182986 80.23 TPP000182988 70.59 TPP000182989 76.75 TPP000182990 78.66 TPP000182991 77.21 TPP000182992 76.2 TPP000182993 65.74 TPP000182994 75.82 TPP000182995 78.92 TPP000182996 76.59 TPP000182997 71.78 TPP000182021 79.12 TPP000161222 38.76

The original molecule was judged to have a purity of 38% by aSEC after protein A purification (TPP000161222), and showed prominent high-molecular species bands in SDS-PAGE gels ( Figure 2A to Figure 2B ). In contrast, the newly designed trimers showed a purity range of 65 to 81%, as judged by aSEC ( Table 1 ). The trimer was further refined using size sieving chromatography, where TPP000161222 showed 98% monodispersity as measured by analytical ultra-centrifugation (AUC), with less than 2% undesired species including high and low molecular weight species ( Figure 3 ).

建構體序列： 形式方案：CD40L-連接子-CD40L-連接子-CD40L- 繫鏈 -人類IgG Fc TPP000183311|OVTSB10|重_鏈| hsctCD40LG2_EKWv2NPQ (SEQ ID NO:1) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL VERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ＞TPP000183310| OVTSB9|heavy_chain|hsctCD40LG2_EKWv2NPQ_G4S3 (SEQ ID NO : 2) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGQVFELQPGASVFLLVNVGTPS GKSSGSGS QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLWLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGGSGGGGSGGGGS VERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ＞TPP000182997|C4LW30|成熟_蛋白| NPQ_G3S3_sthCD40L_G4S2_IgG1_AAS (SEQ ID NO:3) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNR EASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGGSGGGGS EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ＞TPP000182996|C4LW29|成熟_蛋白| NPQ_G3S3_sthCD40L_G4S3_IgG1_AAS (SEQ ID NO:4) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGGSGGGGSGGGGS EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ＞TPP000182995|C4LW28|成熟_蛋白| NPQ_G3S3_sthCD40L_G4S4_IgG1_AAS (SEQ ID NO:5) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGGSGGGGSGGGGSGGGGS EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ＞TPP000182994|C4LW27|成熟_蛋白| NPQ_G3S3_sthCD40L_G4S_IgG1_AAS (SEQ ID NO:6) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLV TLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGGS EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ＞TPP000182993|C4LW26|成熟_蛋白| NPQ_G3S3_sthCD40L_IgG1_AAS (SEQ ID NO:7) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFE LQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ＞TPP000182992|C4LW25|成熟_蛋白| NPQ_sthCD40L_G4S2_IgG1_AAS (SEQ ID NO:8) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGGSGGGGS EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC VVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ＞TPP000182991|C4LW24|成熟_蛋白| NPQ_sthCD40L_G4S3_IgG1_AAS (SEQ ID NO:9) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGGSGGGGSGGGGSE PKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ＞TPP000182990|C4LW23|成熟_蛋白| NPQ_sthCD40L_G4 S4_IgG1_AAS (SEQ ID NO:10) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPG ASVFVNVTDPSQVSHGTGFTSFGLLKL GGGGSGGGGSGGGGSGGGGS EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ＞TPP000182989|C4LW22|成熟_蛋白| NPQ_sthCD40L_G4S_IgG1_AAS (SEQ ID NO:11) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQL TVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGGS EPKSSDKTHTCPPCPAPEAAGGPSVFL F PPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ＞TPP000182988|C4LW21|成熟_蛋白| NPQ_sthCD40L_IgG1_AAS (SEQ ID NO:12) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSH GTGFTSFGLLKL EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK形式方案：人類 IgG Fc - 繫鏈 CD40L-連接子-CD40L-連接子-CD40L ＞TPP000182986|C4LW19|成熟_蛋白| IgG1_AAS_G4S2_NPQ_sthCD40L (SEQ ID NO:13) HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG GGGGSGGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKP CGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL ＞TPP000182985|C4LW18|成熟_蛋白| IgG1_AAS_G4S3_NPQ_sthCD40L (SEQ ID NO:14) HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG GGGGSGGGGSGGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL ＞TPP000182984|C4LW17|成熟_蛋白| IgG1_AAS_G4S4_NPQ_sthCD40L (SEQ ID NO:15) HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG GGGGSGGGGSGGGGSGGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL ＞TPP000182983|C4LW16|成熟_蛋白| IgG1_AAS_G4S_NPQ_G3S3_sthCD40L (SEQ ID NO:16) HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG GGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVN VTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL ＞TPP000182982|C4LW15|成熟_蛋白| IgG1_AAS_G4S2_NPQ_G3S3_sthCD40L (SEQ ID NO:17) HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG GGGGSGGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENG KQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL ＞TPP000182981|C4LW14|成熟_蛋白| IgG1_AAS_G4S3_NPQ_G3S3_sthCD40L (SEQ ID NO:18) HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG GGGGSGGGGSGGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL ＞TPP000182980|C4LW13|成熟_蛋白| IgG1_AAS_G4S4_NPQ_G3S3_sthCD40L (SEQ ID NO:19) HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG GGGGSGGGGSGGGGSGGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL GGGSGGGSGGGS NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL ＞TPP000161222|C40W33|成熟_蛋白| JNJ75347415 (SEQ ID NO:32) IAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL EGKSSGSGS QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGL LKL EGKSSGSGS QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL VERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK實例3：HEK-BLUE檢定

To ensure that the molecular lines are functionally active CD40 agonists, the trimeric fusion proteins were assayed for their ability to activate native CD40 receptors using the HEK-Blue reporter cell line (Invivogen).

HEK-Blue CD40L cells from Invivogen were cultured in growth medium containing DMEM, 2 mM GlutaMAX, 10% heat-inactivated FBS, 100 ug/mL of Normocin, and 100 ug/mL of Pen-Strep. Prepare cells at a concentration of 2.8 x ¹⁰⁵ cells/ml in assay medium (DMEM + 10% heat-inactivated FBS + 100ug/mL PenStrep) for the assay setup. Add 180 µl of the cell suspension to a 96-well plate at a final density of 5 × ¹⁰⁴ cells/well. Add 20 uL of test article to each well for a total volume of 200 µL per well. Incubate the discs at 37 °C in a _CO incubator for 20 to 24 h. The QUANTI-Blue solution from Invivogen was prepared according to the manufacturer's protocol, and 180 µl of the solution was added to each well of a 96-well flat bottom plate. Add 20 uL of primed HEK-Blue CD40L cell supernatant to the assay plate and incubate at 37°C. SEAP levels were determined using a spectrophotometer (635 nm). Figure 5A shows the functional activity of CD40L trimers tested using the HEK-Blue reporter assay. Figure 5B shows the results of a monocyte-derived dendritic cell activation assay. Figure 5C shows the results of an activation assay of mature dendritic cells without cross-linking antibody treatment.

TPP000182983 showed a 100-fold increase in activity when compared to the original molecule TPP000161222, as assayed using a HEK-Blue reporter ( FIG. 5A ). Example 4: MSD Electrochemiluminescence Detection Assay

Binding of the fusion protein to the recombinant CD40 receptor was measured using a Meso Scale Discovery bioluminescent assay (MSD) assay to compare the binding properties of the engineered molecules.

Streptavidin sensor plates were coated with 10 ug/ml of biotinylated CD40 overnight at 4°C. Plates were washed 3X with PBS with 0.05% Tween 20 (PBST). A three-fold dilution series of test articles was prepared in PBS with 0.2% bovine serum albumin and 50ul was applied to the plate for 1 hour at RT with shaking. Plates were washed 3X with PBST and anti-human Fc detection reagent was applied at a final concentration of 2ug/ml for 1 hour at RT with shaking. Plates were washed 3X with PBST and 150ul of 1X read buffer was added to the plates before data was collected on the MSD instrument. Data were imported into GRAPHPAD Prism 8 software and analyzed and graphed based on three separate experiments.

Using MSD and SPR (described in Examples 4 and 5), the newly designed trimers showed increased binding compared to the original designed trimers ( Figure 4A -4C ). Example 5: Surface Plasmon Resonance Analysis of CD40L

Binding of the fusion protein to the recombinant CD40 receptor was measured using a surface plasmon resonance (SPR) assay to compare the binding properties of the engineered molecules.

Surface plasmon resonance (SPR) using Biacore technology requires an interactant to be immobilized or trapped on the sensor surface. Other interactions flow through the modified sensor surface. Changes in the instrument's response are measured to monitor in real time the interaction of two proteins occurring on the sensor surface. SPR experiments for CD40L were performed using a Biacore 8K optical biosensor (GE Healthcare). Samples were prepared in HBS buffer containing 0.05% P20 and experiments were performed at 25°C using a C1 sensor wafer. The wafer was pretreated with 100 mM Gly pH 12-0.3% Triton, which was followed by covalent immobilization of goat anti-human Fc antibody. Goat anti-human IgG Fcγ fragment-specific antibody was diluted in 10 mM sodium acetate buffer (pH 4.5) and ~600 reaction units (RU) were coupled to carboxymethylated dextran on a C1 chip using amine coupling chemistry ( carboxymethylated dextran) surface. The remaining reactive groups on the surface were deactivated using ethanolamine-HCl. For binding experiments, >50 response units (RU) of the CD40L-Fc fusion construct were captured, followed by injection of huCD40 at concentrations ranging from 4.7 nM to 300 nM at 50 µL/min. The association phase was monitored for 3 minutes and the dissociated phase was monitored for 10 minutes. 0.85% H ₃ PO ₄ was injected to regenerate the sensor wafer surface. A flow cell without the CD40L construct was used as a reference. Data were processed using Insight software version 2 (GE healthcare). A double-reference subtraction of the data was performed to correct for buffer contributions to signal and instrument noise.

Interestingly, using both MSD and SPR, the newly designed trimers showed increased binding compared to the original designed trimers ( Figure 4A -4C ). Example 6: CDc+ Dendritic Cell Activation Assay

To assess the ability of trimer fusions to activate human-derived immune cells, a CMV recall assay (Example 7 below) and a dendritic cell cross-presentation assay were employed.

Pre-sorted CD1c+ dendritic cells were thawed, and then 2 × 10 ⁶ cells/mL in complete RPMI 1640 (10% FBS, L-glutamine, non-essential amino acids, sodium pyruvate, and pen/strep) were incubated overnight in a 37°C incubator. The next day, the cells were washed with complete RPMI and seeded in 96-well round-bottom dishes at 1 × ¹⁰⁵ cells/well. Dendritic cells were activated with titrated concentrations of selected CD40 agonist molecules and incubated at 37°C for 24 hours. Cells were washed and subjected to flow cytometric analysis for markers of extracellular dendritic cell activation.

The relative shift of cell surface markers (mainly CD80 and CD86) was measured in the presence and absence of the trimeric fusion protein. Unexpectedly, TPP000182983 showed increased DC activation and T cell activation when compared to other known CD40L agonist trimers (Example 7 below) ( Figure 6A -6E , and Figure 7A -7B ) .

To test the hypothesis that the CD40L trimeric fusion protein is active by itself (i.e., forms a dimer with two copies of the CD40L trimeric fusion protein without interaction between its Fc parts), designs and constructs containing Molecules of CD40L trimers fused to Fc monomeric peptides with mutations in the CH3 domain that prevent dimer formation (“single Fc” molecules, see e.g. 1B ). These single Fc molecules were tested in a 24 hour human DC activation assay with two independent donors. Upregulation of CD86, a marker of APC activation, was observed at levels similar to those of wild-type CD40L (data not shown), suggesting that CD40L trimers alone are active. Example 7: T cell activation assay using monocyte-derived dendritic cells

The presorted mononuclear spheres were thawed and then cultured at 2 × ¹⁰⁶ cells/mL in complete RPMI 1640 (10% FBS, L -Glutamine, non-essential amino acids, sodium pyruvate, and pen/strep) were differentiated into monocyte-derived dendritic cells in a 37°C incubator for 5 days. On day 3, an equal volume of complete RPMI containing 160 ng/mL GM-CSF and 160 ng/mL IL-4 was added. On day 5, cells were washed with complete RPMI and seeded at 3 × ¹⁰⁴ cells/well in 96-well round bottom dishes. To assess T cell activation using monocyte-derived DCs, add titrated amounts of the selected CD40 agonist molecule, 3 x ¹⁰⁵ matched donor T cells, at a final concentration of 1 ug/mL of CEF peptide (CMV, Estin-Barr virus, and influenza virus peptides; ImmunoSpot), and IL-15 (PeproTech, Inc) at a final concentration of 10 µg/mL. On day 2, half of the medium was replaced with complete RPMI containing 20 µg/mL IL-15 and 20 IU/mL IL-2 (R&D Systems). On day 5, half of the medium was replaced with complete RPMI containing 20 ug/mL IL-15, 20 IU/mL IL-2, CEF peptide at a final concentration of 1 ug/mL, CD107a to a final concentration of 1:200 Antibodies (Biolegend), and IX Protein Trafficking Inhibitor Cocktail (Thermo Fisher). Cells were then incubated overnight in a 37°C incubator before proceeding to flow cytometric analysis of intracellular effector interleukin production.

The examples collectively demonstrate that the recently optimized CD40L trimer (TPP000182983) is a stable and highly functional CD40 agonist. Example 8: Analytical Ultracentrifugation

Samples were loaded into centrifuge tanks equipped with 1.2 cm Beckman centerpieces (rated at 50K rpm) and quartz windows. Assemble the tanks and torque them to 130 lb. Place the centrifuge tank in an An-50 (8-hole) or An-60 (4-hole) rotor and place in the Beckman Optima AUC chamber. The temperature of the AUC was equilibrated to 20.5 °C during at least one hour prior to the start of the run (with the rotor tethered in the chamber). mAb samples were run at 40K rpm with scan count (250 scans), scan collection frequency (90 seconds), and data resolution (10 µM). Absorbance data were collected at 280 nm. Initially, the data were analyzed using the software program DCDT (Philo 2006) to determine the location of the meniscus and to observe the profile of the depositional distribution. Next, the data were analyzed using direct boundary fitting software (Stafford and Sherwood, Biophys Chem 108(1-3): 231-243 (2004)). Determine the position of the meniscus by DCDT+, set the baseline at 7.2, and select the fitting range manually. The data were fitted using a non-interaction model of the two species, where the first species corresponds to a monomer and the second species corresponds to a dimer. Example 9: Antitumor Activity

To capture the in vivo antitumor activity of the engineered molecule, colon adenocarcinoma tumor growth was assessed in a human CD40 knock-in mouse model treated with the engineered molecule TPP000182983 (C4LW16). Doses and regimens for TPP000182983 were selected to mimic the expression of encoded proteins from gene delivery platforms (e.g., in the form encoded by mRNA, adeno-associated virus, or oncolytic virus, etc.), which were considered next-best (compared to in recombinant protein therapy).

Human CD40 knock-in mice were implanted with 5 × 10 ⁵ MC38 5AG cells. When the tumor volume reached about 100 mm ³ , the mice were randomly divided into treatment group and control group. Mice were then administered 20 µg of TPP000182983 or a negative control isotype antibody intravenously on days 1, 4, and 6 (after randomization), and tumor growth was measured over time.

During the treatment period, TPP000182983 showed strong antitumor activity with 49.99% inhibition of tumor growth (compared to isotype control) with a p-value of 0.036 (data not shown).

These results demonstrate that engineered trimeric CD40L fusion proteins are effective in inhibiting tumor growth even at suboptimally low doses. Example 10: Virus removal

The ability of engineered molecules to enhance viral clearance was assessed in a human CD40 knock-in mouse model. A group of 6- to 12-week-old female mice was given an intravenous injection of 1 x ¹⁰⁷ pfu of influenza virus through the tail vein. The inoculated mice were randomly divided into treatment group and control group. Then, the treated group of mice was intravenously infused with the polypeptide comprising the single-chain trimeric CD40L fusion protein according to the present disclosure. Control mice were infused with PBS or a negative control isotype antibody. Virus titers in mice were measured over time.

During treatment, mice administered the engineered trimeric CD40L fusion protein had lower virus titers and faster viral clearance compared to mice administered negative control isotype antibody or PBS.

These results are to demonstrate that engineered trimeric CD40L fusion proteins according to the present disclosure are effective in enhancing influenza virus clearance. Example 11: Vaccine Adjuvants

Engineered trimeric CD40L fusion proteins were evaluated for their ability to enhance immune responses to vaccine compositions in human CD40 knock-in mice.

Human CD40 knock-in mice were randomly divided into groups, and a vaccine composition including the target virus antigen was administered to them. The polypeptide comprising the single-chain trimeric CD40L fusion protein according to the present disclosure was administered to the mice simultaneously or sequentially with the vaccine composition (treatment group). In the control group, mice were administered PBS (instead of the engineered trimeric CD40L fusion protein). The immune response to viral antigens in the treated and control groups of mice was measured over time. The results showed that mice in the treatment group exhibited significantly higher immune responses to viral antigens than mice in the control group. Specifically, the titers of antibodies specifically binding to the target viral antigen were significantly higher, and the concentration of antibody-producing plasma cells was significantly higher (in the treated mice), The above is compared with the control group.

Treated and control mice were sacrificed at the end of the observation period, and anatomical tissues known to be infected with the virus (eg, lungs) were isolated and prepared for further analysis. In in situ immunohistochemical studies, Alexa™ Fluor 488 is used to stain dendritic cells for cell-specific markers, allowing dendritic cells in tissue samples to appear as green dots under fluorescence microscopy. The viral antigens of interest used in the vaccine composition are stained with Alexa™ Fluor 568 to visualize the antigen molecules as red dots. Dendritic cell lines presenting viral antigens were visualized by co-localization of the two fluorescent signals. Using fluorescence microscopy, the total number of dendritic cells and the percentage of dendritic cells presenting viral antigens in tissue samples were measured. The results showed a significant percentage of mature dendritic cells presenting viral antigens in mice that received the vaccine composition together with the engineered trimeric CD40L fusion protein (treatment group) compared to mice that received the vaccine composition alone (control group) Increase.

To further assess the effect of engineered trimeric CD40L fusion proteins on viral antigen presentation by antigen presenting cells, immunoprecipitation studies were performed to measure the association between viral antigens and MHC complexes. Homogenates of dissected lung tissue from sacrificed mice were prepared according to standard protocols. Under suitable conditions, the homogenate is incubated with beads (which are coated with monoclonal antibodies that specifically bind to MHC class I or MHC class II molecules) to allow the antibody to interact with its target protein. and protein complexes (in tissue samples). The beads are then separated from the tissue by centrifugation and the proteins and protein complexes associated with the coated antibodies are pulled down. Then, pull down protein samples were processed for analysis of their contents via Western blot. Monoclonal antibodies specific to the viral antigen of interest are used. The results showed that MHC molecules (the study The amount of viral antigen immunoprecipitated by both class I and class II molecules examined in the study was significantly increased.

These results are to demonstrate that the engineered trimeric CD40L fusion protein according to the present disclosure is effective in enhancing the immune response to the vaccine when co-administered with the vaccine in an adjuvant. The increased immune response is attributable to at least increased viral antigen presentation by antigen presenting cells, such as dendritic cells, in the immunized animal.

To monitor the effects of engineered trimeric CD40L fusion proteins on long-term immune memory, mice were administered an initial dose of a vaccine composition in combination with a polypeptide comprising a trimeric CD40L fusion protein according to the present disclosure (therapeutic Group). The mice were then divided into a first group of supplemented mice and a second group of non-supplemented mice, wherein the supplemented mice were given a second dose of the vaccine composition on day 7, and the non-supplemented mice were given a second dose on day 7. Mouse PBS. In the control group, mice were administered the vaccine composition alone on day 0, and the mice were divided into booster and non-booster groups to receive a second dose of the vaccine composition or PBS on day 7, respectively . The immune response to viral antigens in the treated and control groups of mice was measured over time.

The results are to show that mice in both the treatment group and the control group, after receiving the initial dose of the vaccine composition, develop antibodies that specifically bind to the target viral antigen. On the 14th and 28th day of the treatment group, the antibody titer in the non-supplemented mice was still high, and was comparable to that in the supplemented mice. In the control group mice, the antibody titer in the non-supplemented mice was significantly lower than that in the supplemented mice on day 28. Mice were sacrificed on day 28, while control mice were observed to develop memory B cells, but no memory B cell formation was observed in control mice (boosted or not).

These results are intended to indicate that the engineered trimeric CD40L fusion protein according to the present disclosure effectively enhances the humoral immune response and antibody production targeting viral antigens, and promotes faster formation of immune memory against the virus. Engineered trimeric CD40L fusion proteins according to the present disclosure can be used as adjuvants in vaccine compositions specifically adapted to prevent infection by a virus of interest in a subject. Example 12: FC monomer peptide activity

Fc monomer peptides were designed to test the hypothesis that Fc monomers are active. Fc monomers were tested in a 24 hour human DC activation assay with two independent donors. Similar to WT CD40L activity, CD86 was upregulated (which is a marker of APC activation), indicating that the Fc monomer is active. Example 13: Prognostic Methods for Determining Treatment Suitability

Individuals or subpopulations of cancer or solid tumor patients who would benefit from treatment with a single-chain trimeric CD40L fusion protein according to the present disclosure can be identified using a prognostic assay that looks at the single-chain trimeric CD40L fusion protein administered Prognostic markers are present or absent, or their levels are increased or decreased. Subjects identified as responding to treatment with a single-chain trimeric CD40L fusion protein according to the present disclosure may thus receive treatment. Instead, they may use an ex vivo prognostic assay to further screen for the presence of a single-chain trimeric CD40L fusion protein. Potential benefits, eg, in identifying whether a cancer or solid tumor would be more responsive to a treatment comprising administration of a single chain trimeric CD40L fusion protein according to the present disclosure.

The ex vivo prognostic assay comprises providing a test biological sample and a control biological sample (from a candidate subject), wherein the biological samples comprise CD40-expressing antigen-presenting cells that have been in vivo expressed with a single-chain trimeric CD40L fusion protein or ex vivo stimulation; detecting the expression level of at least one biomarker in the test biological sample; and detecting the expression level of the biomarker(s) in a control biological sample (not yet in contact with the single-chain trimeric CD40L fusion protein). The corresponding performance levels measured were compared. Biomarkers used in this in vivo prognostic assay include proteins and/or genes whose expression levels are prognostic indicators of response to therapeutic intervention. * * * * *

Those of ordinary skill in the art will appreciate that changes can be made to the above-described embodiments without departing from their broad inventive concepts. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention, as defined by this description.

The foregoing summary of the invention and the following implementation descriptions of the specific embodiments of the application will be better understood when read in conjunction with the accompanying drawings. It should be understood, however, that the application is not limited to the precise embodiments shown in the drawings. [ FIG. 1A ] to [ FIG. 1B ] schematic diagrams showing examples of trimeric CD40L Fc fusion proteins. Figure 1A shows a schematic of CD40L trimer design optimization in which the molecule was designed to manipulate tether length, linker length, and fusion orientation. Figure 1B shows a schematic representation of a "single Fc" molecule, which is a single-chain fusion protein containing a CD40L trimer (circled) fused to an Fc monomer Peptides wherein the Fc contains mutations in its CH3 domain that prevent dimer formation of this molecule. [ FIG. 2A ] to [ FIG. 2B ] show the monodispersity of CD40L trimers on Fc after protein A purification. Figure 2A shows the SDS-PAGE gel (left panel) and SEC chromatogram (right panel) of TPP000161222, and Figure 2B shows the SDS-PAGE gel and SEC chromatogram of TPP000182983. [ FIG. 3 ] shows the monodispersity of TPP000182983 after size sizing chromatography, which was measured via analytical ultra-centrifugation (AUC). All samples were analyzed in duplicate (n=2). Proteins run mostly at ~7 s, with 98% of the major species groups. <1% lower molecular weight species (LMWS), <2% dimers, and <1% high molecular weight species (HMWS) were also present. [ FIG. 4A ] to [ FIG. 4C ] show the binding of CD40 agonists to CD40. Figure 4A shows the binding of CD40L trimers to CD40R as measured using a Meso Scale Discovery (MSD) bioluminescence assay. Figure 4B shows the binding of TPP000182983 to CD40R as measured using surface plasmon resonance (SPR). Figure 4C shows the binding of TPP000161222 to CD40R as measured using surface plasmon resonance (SPR). [ FIG. 5A ] to [ FIG. 5C ] show the functional activity of CD40L trimers using the following assays: HEK-Blue reporter assay of FIG. 5A , monocyte-derived dendritic cell activation assay of FIG. 5B , and Mature dendritic cell activation assay (untreated with cross-linked antibody). [ FIG. 6A ] to [ FIG. 6E ] show the assessment of CD40L trimeric Fc functional activity in a dendritic cell activation assay using the D86 activation marker ( FIG. 6A ); the CD40 activation marker ( FIG. 6B ); the CD83 activation marker ( Figure 6C); HLA-DR activation markers (Figure 6D) and PDL1 activation markers (Figure 6E). [ FIG. 7A ] to [ FIG. 7B ] show the following T cell responses generated from moDCs in the presence of CD40 agonist and CEF peptide: CD8+ T cells ( FIG. 7A ) and CD4+ T cells ( FIG. 7B ).

         <![CDATA[ <110> Janssen Biotech, Inc.]]>
           <![CDATA[ <120> Bioengineered immunomodulatory fusion protein composition]]>
           <![CDATA[ <130> 14620-628-228 / JBI6455WOPCT1]]>
           <![CDATA[ <140> TW 111109113]]>
           <![CDATA[ <141> 2022-03-11]]>
           <![CDATA[ <150> US 63/160,694]]>
           <![CDATA[ <151> 2021-03-12]]>
           <![CDATA[ <150> US 63/160,693]]>
           <![CDATA[ <151> 2021-03-12]]>
           <![CDATA[ <150> US 63/160,691]]>
           <![CDATA[ <151> 2021-03-12]]>
           <![CDATA[ <150> US 63/160,688]]>
           <![CDATA[ <151> 2021-03-12]]>
           <![CDATA[ <150> US 63/160,686]]>
           <![CDATA[ <151> 2021-03-12]]>
           <![CDATA[ <160> 40 ]]>
           <![CDATA[ <170> PatentIn Version 3.5]]>
           <![CDATA[ <210> 1]]>
           <![CDATA[ <211> 672]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000183311 - OVTSB10 - heavy_chain - hsctCD40LG2_EKWv2NPQ]]>
           <![CDATA[ <400> 1]]>
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          1 5 10 15
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                      20 25 30
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                  35 40 45
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
              50 55 60
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Trp Leu Lys Ser Pro
          65 70 75 80
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
                          85 90 95
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                      100 105 110
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                  115 120 125
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Glu
              130 135 140
          Gly Lys Ser Ser Gly Ser Gly Ser Gln Ile Ala Ala His Val Ile Ser
          145 150 155 160
          Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
                          165 170 175
          Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln
                      180 185 190
          Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr
                  195 200 205
          Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser
              210 215 220
          Leu Trp Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala
          225 230 235 240
          Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His
                          245 250 255
          Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn
                      260 265 270
          Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
                  275 280 285
          Gly Leu Leu Lys Leu Glu Gly Lys Ser Ser Gly Ser Gly Ser Gln Ile
              290 295 300
          Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu
          305 310 315 320
          Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr
                          325 330 335
          Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr
                      340 345 350
          Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Ser Gln
                  355 360 365
          Ala Pro Phe Ile Ala Ser Leu Trp Leu Lys Ser Pro Gly Arg Phe Glu
              370 375 380
          Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys
          385 390 395 400
          Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly
                          405 410 415
          Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly
                      420 425 430
          Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Val Glu Arg Lys Ser
                  435 440 445
          Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser
              450 455 460
          Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
          465 470 475 480
          Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
                          485 490 495
          Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
                      500 505 510
          Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val
                  515 520 525
          Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
              530 535 540
          Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr
          545 550 555 560
          Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
                          565 570 575
          Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
                      580 585 590
          Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
                  595 600 605
          Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp
              610 615 620
          Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
          625 630 635 640
          Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
                          645 650 655
          Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
                      660 665 670
           <![CDATA[ <210> 2]]>
           <![CDATA[ <211> 687]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000183310 - OVTSB9 - heavy_chain - hsctCD40LG2_EKWv2NPQ_G4S3]]>
           <![CDATA[ <400> 2]]>
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          1 5 10 15
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                      20 25 30
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                  35 40 45
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
              50 55 60
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Trp Leu Lys Ser Pro
          65 70 75 80
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
                          85 90 95
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                      100 105 110
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                  115 120 125
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Glu
              130 135 140
          Gly Lys Ser Ser Gly Ser Gly Ser Gln Ile Ala Ala His Val Ile Ser
          145 150 155 160
          Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
                          165 170 175
          Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln
                      180 185 190
          Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr
                  195 200 205
          Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser
              210 215 220
          Leu Trp Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala
          225 230 235 240
          Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His
                          245 250 255
          Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn
                      260 265 270
          Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
                  275 280 285
          Gly Leu Leu Lys Leu Glu Gly Lys Ser Ser Gly Ser Gly Ser Gln Ile
              290 295 300
          Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu
          305 310 315 320
          Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr
                          325 330 335
          Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr
                      340 345 350
          Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Ser Gln
                  355 360 365
          Ala Pro Phe Ile Ala Ser Leu Trp Leu Lys Ser Pro Gly Arg Phe Glu
              370 375 380
          Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys
          385 390 395 400
          Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly
                          405 410 415
          Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly
                      420 425 430
          Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly Gly Ser
                  435 440 445
          Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Glu Arg Lys Ser Cys
              450 455 460
          Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val
          465 470 475 480
          Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
                          485 490 495
          Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
                      500 505 510
          Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
                  515 520 525
          Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser
              530 535 540
          Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
          545 550 555 560
          Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile
                          565 570 575
          Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
                      580 585 590
          Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
                  595 600 605
          Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
              610 615 620
          Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
          625 630 635 640
          Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
                          645 650 655
          Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
                      660 665 670
          His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
                  675 680 685
           <![CDATA[ <210> 3]]>
           <![CDATA[ <211> 695]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000182997 - C4LW30 - mature_protein - ]]>
                 NPQ_G3S3_sthCD40L_G4S2_IgG1_AAS
           <![CDATA[ <400> 3]]>
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          1 5 10 15
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                      20 25 30
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                  35 40 45
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
              50 55 60
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
          65 70 75 80
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
                          85 90 95
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                      100 105 110
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                  115 120 125
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly
              130 135 140
          Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Asn Pro Gln Ile Ala
          145 150 155 160
          Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln
                          165 170 175
          Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Asn Leu Val Thr Leu
                      180 185 190
          Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile
                  195 200 205
          Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Ser Gln Ala
              210 215 220
          Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg
          225 230 235 240
          Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly
                          245 250 255
          Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala
                      260 265 270
          Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr
                  275 280 285
          Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly Ser Gly Gly
              290 295 300
          Gly Ser Gly Gly Gly Ser Asn Pro Gln Ile Ala Ala His Val Ile Ser
          305 310 315 320
          Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
                          325 330 335
          Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln
                      340 345 350
          Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr
                  355 360 365
          Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser
              370 375 380
          Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala
          385 390 395 400
          Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His
                          405 410 415
          Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn
                      420 425 430
          Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
                  435 440 445
          Gly Leu Leu Lys Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu
              450 455 460
          Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
          465 470 475 480
          Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
                          485 490 495
          Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
                      500 505 510
          Ser Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
                  515 520 525
          Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
              530 535 540
          Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
          545 550 555 560
          Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
                          565 570 575
          Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
                      580 585 590
          Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
                  595 600 605
          Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
              610 615 620
          Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
          625 630 635 640
          Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
                          645 650 655
          Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
                      660 665 670
          Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
                  675 680 685
          Leu Ser Leu Ser Pro Gly Lys
              690 695
           <![CDATA[ <210> 4]]>
           <![CDATA[ <211> 700]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000182996 - C4LW29 - mature_protein - ]]>
                 NPQ_G3S3_sthCD40L_G4S3_IgG1_AAS
           <![CDATA[ <400> 4]]>
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          1 5 10 15
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                      20 25 30
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                  35 40 45
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
              50 55 60
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
          65 70 75 80
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
                          85 90 95
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                      100 105 110
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                  115 120 125
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly
              130 135 140
          Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Asn Pro Gln Ile Ala
          145 150 155 160
          Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln
                          165 170 175
          Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Asn Leu Val Thr Leu
                      180 185 190
          Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile
                  195 200 205
          Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Ser Gln Ala
              210 215 220
          Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg
          225 230 235 240
          Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly
                          245 250 255
          Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala
                      260 265 270
          Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr
                  275 280 285
          Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly Ser Gly Gly
              290 295 300
          Gly Ser Gly Gly Gly Ser Asn Pro Gln Ile Ala Ala His Val Ile Ser
          305 310 315 320
          Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
                          325 330 335
          Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln
                      340 345 350
          Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr
                  355 360 365
          Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser
              370 375 380
          Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala
          385 390 395 400
          Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His
                          405 410 415
          Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn
                      420 425 430
          Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
                  435 440 445
          Gly Leu Leu Lys Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
              450 455 460
          Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro
          465 470 475 480
          Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
                          485 490 495
          Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
                      500 505 510
          Thr Cys Val Val Val Ser Val Ser His Glu Asp Pro Glu Val Lys Phe
                  515 520 525
          Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
              530 535 540
          Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
          545 550 555 560
          Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
                          565 570 575
          Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
                      580 585 590
          Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
                  595 600 605
          Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
              610 615 620
          Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
          625 630 635 640
          Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
                          645 650 655
          Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
                      660 665 670
          Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
                  675 680 685
          Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
              690 695 700
           <![CDATA[ <210> 5]]>
           <![CDATA[ <211> 705]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000182995 - C4LW28 - mature_protein - ]]>
                 NPQ_G3S3_sthCD40L_G4S4_IgG1_AAS
           <![CDATA[ <400> 5]]>
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          1 5 10 15
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                      20 25 30
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                  35 40 45
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
              50 55 60
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
          65 70 75 80
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
                          85 90 95
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                      100 105 110
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                  115 120 125
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly
              130 135 140
          Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Asn Pro Gln Ile Ala
          145 150 155 160
          Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln
                          165 170 175
          Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Asn Leu Val Thr Leu
                      180 185 190
          Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile
                  195 200 205
          Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Ser Gln Ala
              210 215 220
          Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg
          225 230 235 240
          Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly
                          245 250 255
          Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala
                      260 265 270
          Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr
                  275 280 285
          Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly Ser Gly Gly
              290 295 300
          Gly Ser Gly Gly Gly Ser Asn Pro Gln Ile Ala Ala His Val Ile Ser
          305 310 315 320
          Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
                          325 330 335
          Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln
                      340 345 350
          Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr
                  355 360 365
          Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser
              370 375 380
          Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala
          385 390 395 400
          Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His
                          405 410 415
          Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn
                      420 425 430
          Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
                  435 440 445
          Gly Leu Leu Lys Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
              450 455 460
          Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys
          465 470 475 480
          Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
                          485 490 495
          Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
                      500 505 510
          Arg Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser His Glu Asp
                  515 520 525
          Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
              530 535 540
          Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
          545 550 555 560
          Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
                          565 570 575
          Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
                      580 585 590
          Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
                  595 600 605
          Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
              610 615 620
          Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
          625 630 635 640
          Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
                          645 650 655
          Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
                      660 665 670
          Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
                  675 680 685
          Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
              690 695 700
          Lys
          705
           <![CDATA[ <210> 6]]>
           <![CDATA[ <211> 690]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000182994 - C4LW27 - mature_protein - ]]>
                 NPQ_G3S3_sthCD40L_G4S_IgG1_AAS
           <![CDATA[ <400> 6]]>
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          1 5 10 15
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                      20 25 30
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                  35 40 45
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
              50 55 60
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
          65 70 75 80
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
                          85 90 95
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                      100 105 110
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                  115 120 125
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly
              130 135 140
          Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Asn Pro Gln Ile Ala
          145 150 155 160
          Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln
                          165 170 175
          Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Asn Leu Val Thr Leu
                      180 185 190
          Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile
                  195 200 205
          Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Ser Gln Ala
              210 215 220
          Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg
          225 230 235 240
          Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly
                          245 250 255
          Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala
                      260 265 270
          Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr
                  275 280 285
          Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly Ser Gly Gly
              290 295 300
          Gly Ser Gly Gly Gly Ser Asn Pro Gln Ile Ala Ala His Val Ile Ser
          305 310 315 320
          Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
                          325 330 335
          Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln
                      340 345 350
          Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr
                  355 360 365
          Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser
              370 375 380
          Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala
          385 390 395 400
          Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His
                          405 410 415
          Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn
                      420 425 430
          Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
                  435 440 445
          Gly Leu Leu Lys Leu Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp
              450 455 460
          Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly
          465 470 475 480
          Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
                          485 490 495
          Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser His Glu
                      500 505 510
          Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
                  515 520 525
          Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
              530 535 540
          Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
          545 550 555 560
          Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
                          565 570 575
          Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
                      580 585 590
          Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
                  595 600 605
          Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
              610 615 620
          Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
          625 630 635 640
          Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
                          645 650 655
          Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
                      660 665 670
          Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
                  675 680 685
          Gly Lys
              690
           <![CDATA[ <210> 7]]>
           <![CDATA[ <211> 685]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000182993 - C4LW26 - mature_protein - ]]>
                 NPQ_G3S3_sthCD40L_IgG1_AAS
           <![CDATA[ <400> 7]]>
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          1 5 10 15
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                      20 25 30
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                  35 40 45
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
              50 55 60
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
          65 70 75 80
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
                          85 90 95
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                      100 105 110
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                  115 120 125
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly
              130 135 140
          Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Asn Pro Gln Ile Ala
          145 150 155 160
          Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln
                          165 170 175
          Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Asn Leu Val Thr Leu
                      180 185 190
          Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile
                  195 200 205
          Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Ser Gln Ala
              210 215 220
          Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg
          225 230 235 240
          Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly
                          245 250 255
          Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala
                      260 265 270
          Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr
                  275 280 285
          Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly Ser Gly Gly
              290 295 300
          Gly Ser Gly Gly Gly Ser Asn Pro Gln Ile Ala Ala His Val Ile Ser
          305 310 315 320
          Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
                          325 330 335
          Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln
                      340 345 350
          Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr
                  355 360 365
          Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser
              370 375 380
          Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala
          385 390 395 400
          Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His
                          405 410 415
          Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn
                      420 425 430
          Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
                  435 440 445
          Gly Leu Leu Lys Leu Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys
              450 455 460
          Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu
          465 470 475 480
          Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
                          485 490 495
          Val Thr Cys Val Val Val Ser Val Ser His Glu Asp Pro Glu Val Lys
                      500 505 510
          Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
                  515 520 525
          Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
              530 535 540
          Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
          545 550 555 560
          Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
                          565 570 575
          Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
                      580 585 590
          Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
                  595 600 605
          Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
              610 615 620
          Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
          625 630 635 640
          Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
                          645 650 655
          Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
                      660 665 670
          His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
                  675 680 685
           <![CDATA[ <210> 8]]>
           <![CDATA[ <211> 689]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000182992 - C4LW25 - mature_protein - ]]>
                 NPQ_sthCD40L_G4S2_IgG1_AAS
           <![CDATA[ <400> 8]]>
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          1 5 10 15
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                      20 25 30
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                  35 40 45
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
              50 55 60
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
          65 70 75 80
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
                          85 90 95
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                      100 105 110
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                  115 120 125
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Glu
              130 135 140
          Gly Lys Ser Ser Gly Ser Gly Ser Asn Pro Gln Ile Ala Ala His Val
          145 150 155 160
          Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu
                          165 170 175
          Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly
                      180 185 190
          Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln
                  195 200 205
          Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile
              210 215 220
          Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu
          225 230 235 240
          Arg Ala Ala Asn Thr His Ser Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser
                          245 250 255
          Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe
                      260 265 270
          Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr
                  275 280 285
          Ser Phe Gly Leu Leu Lys Leu Glu Gly Lys Ser Ser Gly Ser Gly Ser
              290 295 300
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          305 310 315 320
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                          325 330 335
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                      340 345 350
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
                  355 360 365
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
              370 375 380
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
          385 390 395 400
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                          405 410 415
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                      420 425 430
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly
                  435 440 445
          Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys
              450 455 460
          Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro
          465 470 475 480
          Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
                          485 490 495
          Arg Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser His Glu Asp
                      500 505 510
          Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
                  515 520 525
          Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
              530 535 540
          Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
          545 550 555 560
          Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
                          565 570 575
          Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
                      580 585 590
          Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
                  595 600 605
          Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
              610 615 620
          Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
          625 630 635 640
          Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
                          645 650 655
          Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
                      660 665 670
          Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
                  675 680 685
          Lys
           <![CDATA[ <210> 9]]>
           <![CDATA[ <211> 694]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000182991 - C4LW24 - mature_protein - ]]>
                 NPQ_sthCD40L_G4S3_IgG1_AAS
           <![CDATA[ <400> 9]]>
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          1 5 10 15
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                      20 25 30
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                  35 40 45
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
              50 55 60
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
          65 70 75 80
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
                          85 90 95
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                      100 105 110
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                  115 120 125
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Glu
              130 135 140
          Gly Lys Ser Ser Gly Ser Gly Ser Asn Pro Gln Ile Ala Ala His Val
          145 150 155 160
          Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu
                          165 170 175
          Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly
                      180 185 190
          Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln
                  195 200 205
          Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile
              210 215 220
          Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu
          225 230 235 240
          Arg Ala Ala Asn Thr His Ser Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser
                          245 250 255
          Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe
                      260 265 270
          Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr
                  275 280 285
          Ser Phe Gly Leu Leu Lys Leu Glu Gly Lys Ser Ser Gly Ser Gly Ser
              290 295 300
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          305 310 315 320
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                          325 330 335
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                      340 345 350
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
                  355 360 365
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
              370 375 380
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
          385 390 395 400
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                          405 410 415
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                      420 425 430
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly
                  435 440 445
          Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro
              450 455 460
          Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
          465 470 475 480
          Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
                          485 490 495
          Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ser
                      500 505 510
          Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
                  515 520 525
          Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
              530 535 540
          Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
          545 550 555 560
          Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
                          565 570 575
          Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
                      580 585 590
          Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
                  595 600 605
          Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
              610 615 620
          Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
          625 630 635 640
          Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
                          645 650 655
          Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
                      660 665 670
          Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
                  675 680 685
          Ser Leu Ser Pro Gly Lys
              690
           <![CDATA[ <210> 10]]>
           <![CDATA[ <211> 699]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000182990 - C4LW23 - mature_protein - ]]>
                 NPQ_sthCD40L_G4S4_IgG1_AAS
           <![CDATA[ <400> 10]]>
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          1 5 10 15
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                      20 25 30
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                  35 40 45
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
              50 55 60
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
          65 70 75 80
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
                          85 90 95
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                      100 105 110
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                  115 120 125
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Glu
              130 135 140
          Gly Lys Ser Ser Gly Ser Gly Ser Asn Pro Gln Ile Ala Ala His Val
          145 150 155 160
          Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu
                          165 170 175
          Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly
                      180 185 190
          Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln
                  195 200 205
          Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile
              210 215 220
          Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu
          225 230 235 240
          Arg Ala Ala Asn Thr His Ser Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser
                          245 250 255
          Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe
                      260 265 270
          Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr
                  275 280 285
          Ser Phe Gly Leu Leu Lys Leu Glu Gly Lys Ser Ser Gly Ser Gly Ser
              290 295 300
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          305 310 315 320
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                          325 330 335
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                      340 345 350
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
                  355 360 365
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
              370 375 380
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
          385 390 395 400
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                          405 410 415
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                      420 425 430
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly
                  435 440 445
          Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
              450 455 460
          Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro
          465 470 475 480
          Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro
                          485 490 495
          Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
                      500 505 510
          Cys Val Val Val Ser Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
                  515 520 525
          Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
              530 535 540
          Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
          545 550 555 560
          Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
                          565 570 575
          Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
                      580 585 590
          Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
                  595 600 605
          Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
              610 615 620
          Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
          625 630 635 640
          Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
                          645 650 655
          Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
                      660 665 670
          Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
                  675 680 685
          Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
              690 695
           <![CDATA[ <210> 11]]>
           <![CDATA[ <211> 684]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000182989 - C4LW22 - mature_protein - ]]>
                 NPQ_sthCD40L_G4S_IgG1_AAS
           <![CDATA[ <400> 11]]>
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          1 5 10 15
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                      20 25 30
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                  35 40 45
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
              50 55 60
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
          65 70 75 80
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
                          85 90 95
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                      100 105 110
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                  115 120 125
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Glu
              130 135 140
          Gly Lys Ser Ser Gly Ser Gly Ser Asn Pro Gln Ile Ala Ala His Val
          145 150 155 160
          Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu
                          165 170 175
          Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly
                      180 185 190
          Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln
                  195 200 205
          Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile
              210 215 220
          Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu
          225 230 235 240
          Arg Ala Ala Asn Thr His Ser Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser
                          245 250 255
          Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe
                      260 265 270
          Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr
                  275 280 285
          Ser Phe Gly Leu Leu Lys Leu Glu Gly Lys Ser Ser Gly Ser Gly Ser
              290 295 300
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          305 310 315 320
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                          325 330 335
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                      340 345 350
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
                  355 360 365
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
              370 375 380
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
          385 390 395 400
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                          405 410 415
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                      420 425 430
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly
                  435 440 445
          Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro
              450 455 460
          Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe
          465 470 475 480
          Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
                          485 490 495
          Thr Cys Val Val Val Ser Val Ser His Glu Asp Pro Glu Val Lys Phe
                      500 505 510
          Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
                  515 520 525
          Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
              530 535 540
          Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
          545 550 555 560
          Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
                          565 570 575
          Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
                      580 585 590
          Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
                  595 600 605
          Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
              610 615 620
          Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
          625 630 635 640
          Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
                          645 650 655
          Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
                      660 665 670
          Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
                  675 680
           <![CDATA[ <210> 12]]>
           <![CDATA[ <211> 679]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000182988 - C4LW21 - mature_protein - NPQ_sthCD40L_IgG1_AAS]]>
           <![CDATA[ <400> 12]]>
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          1 5 10 15
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                      20 25 30
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                  35 40 45
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
              50 55 60
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
          65 70 75 80
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
                          85 90 95
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                      100 105 110
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                  115 120 125
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Glu
              130 135 140
          Gly Lys Ser Ser Gly Ser Gly Ser Asn Pro Gln Ile Ala Ala His Val
          145 150 155 160
          Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu
                          165 170 175
          Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly
                      180 185 190
          Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln
                  195 200 205
          Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile
              210 215 220
          Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu
          225 230 235 240
          Arg Ala Ala Asn Thr His Ser Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser
                          245 250 255
          Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe
                      260 265 270
          Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr
                  275 280 285
          Ser Phe Gly Leu Leu Lys Leu Glu Gly Lys Ser Ser Gly Ser Gly Ser
              290 295 300
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          305 310 315 320
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                          325 330 335
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                      340 345 350
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
                  355 360 365
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
              370 375 380
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
          385 390 395 400
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                          405 410 415
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                      420 425 430
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Glu
                  435 440 445
          Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
              450 455 460
          Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
          465 470 475 480
          Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
                          485 490 495
          Ser Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
                      500 505 510
          Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
                  515 520 525
          Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
              530 535 540
          Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
          545 550 555 560
          Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
                          565 570 575
          Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
                      580 585 590
          Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
                  595 600 605
          Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
              610 615 620
          Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
          625 630 635 640
          Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
                          645 650 655
          Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
                      660 665 670
          Leu Ser Leu Ser Pro Gly Lys
                  675
           <![CDATA[ <210> 13]]>
           <![CDATA[ <211> 680]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000182986 - C4LW19 - mature_protein - ]]>
                 IgG1_AAS_G4S2_NPQ_sthCD40L
           <![CDATA[ <400> 13]]>
          His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
          1 5 10 15
          Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
                      20 25 30
          Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser His Glu Asp Pro
                  35 40 45
          Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
              50 55 60
          Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
          65 70 75 80
          Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
                          85 90 95
          Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
                      100 105 110
          Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
                  115 120 125
          Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
              130 135 140
          Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
          145 150 155 160
          Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
                          165 170 175
          Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
                      180 185 190
          Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
                  195 200 205
          Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly
              210 215 220
          Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Pro Gln Ile Ala Ala His
          225 230 235 240
          Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala
                          245 250 255
          Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn
                      260 265 270
          Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala
                  275 280 285
          Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe
              290 295 300
          Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu
          305 310 315 320
          Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln
                          325 330 335
          Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val
                      340 345 350
          Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe
                  355 360 365
          Thr Ser Phe Gly Leu Leu Lys Leu Glu Gly Lys Ser Ser Gly Ser Gly
              370 375 380
          Ser Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Ser Lys
          385 390 395 400
          Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser
                          405 410 415
          Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg
                      420 425 430
          Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg
                  435 440 445
          Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser
              450 455 460
          Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser
          465 470 475 480
          Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe
                          485 490 495
          Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser
                      500 505 510
          Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu
                  515 520 525
          Glu Gly Lys Ser Ser Ser Gly Ser Gly Ser Asn Pro Gln Ile Ala Ala His
              530 535 540
          Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala
          545 550 555 560
          Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn
                          565 570 575
          Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala
                      580 585 590
          Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe
                  595 600 605
          Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu
              610 615 620
          Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln
          625 630 635 640
          Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val
                          645 650 655
          Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe
                      660 665 670
          Thr Ser Phe Gly Leu Leu Lys Leu
                  675 680
           <![CDATA[ <210> 14]]>
           <![CDATA[ <211> 685]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000182985 - C4LW18 - mature_protein - ]]>
                 IgG1_AAS_G4S3_NPQ_sthCD40L
           <![CDATA[ <400> 14]]>
          His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
          1 5 10 15
          Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
                      20 25 30
          Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser His Glu Asp Pro
                  35 40 45
          Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
              50 55 60
          Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
          65 70 75 80
          Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
                          85 90 95
          Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
                      100 105 110
          Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
                  115 120 125
          Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
              130 135 140
          Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
          145 150 155 160
          Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
                          165 170 175
          Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
                      180 185 190
          Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
                  195 200 205
          Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly
              210 215 220
          Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Pro
          225 230 235 240
          Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser
                          245 250 255
          Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu
                      260 265 270
          Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu
                  275 280 285
          Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser
              290 295 300
          Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg
          305 310 315 320
          Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ser Ala Lys
                          325 330 335
          Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln
                      340 345 350
          Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser
                  355 360 365
          His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Glu Gly Lys
              370 375 380
          Ser Ser Gly Ser Gly Ser Asn Pro Gln Ile Ala Ala His Val Ile Ser
          385 390 395 400
          Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
                          405 410 415
          Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln
                      420 425 430
          Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr
                  435 440 445
          Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser
              450 455 460
          Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala
          465 470 475 480
          Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His
                          485 490 495
          Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn
                      500 505 510
          Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
                  515 520 525
          Gly Leu Leu Lys Leu Glu Gly Lys Ser Ser Gly Ser Gly Ser Asn Pro
              530 535 540
          Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser
          545 550 555 560
          Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu
                          565 570 575
          Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu
                      580 585 590
          Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser
                  595 600 605
          Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg
              610 615 620
          Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ser Ala Lys
          625 630 635 640
          Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln
                          645 650 655
          Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser
                      660 665 670
          His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu
                  675 680 685
           <![CDATA[ <210> 15]]>
           <![CDATA[ <211> 690]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000182984 - C4LW17 - mature_protein - ]]>
                 IgG1_AAS_G4S4_NPQ_sthCD40L
           <![CDATA[ <400> 15]]>
          His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
          1 5 10 15
          Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
                      20 25 30
          Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser His Glu Asp Pro
                  35 40 45
          Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
              50 55 60
          Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
          65 70 75 80
          Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
                          85 90 95
          Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
                      100 105 110
          Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
                  115 120 125
          Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
              130 135 140
          Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
          145 150 155 160
          Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
                          165 170 175
          Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
                      180 185 190
          Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
                  195 200 205
          Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly
              210 215 220
          Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
          225 230 235 240
          Gly Gly Ser Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser
                          245 250 255
          Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr
                      260 265 270
          Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val
                  275 280 285
          Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser
              290 295 300
          Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu
          305 310 315 320
          Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr
                          325 330 335
          His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly
                      340 345 350
          Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp
                  355 360 365
          Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu
              370 375 380
          Lys Leu Glu Gly Lys Ser Ser Gly Ser Gly Ser Asn Pro Gln Ile Ala
          385 390 395 400
          Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln
                          405 410 415
          Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Asn Leu Val Thr Leu
                      420 425 430
          Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile
                  435 440 445
          Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Ser Gln Ala
              450 455 460
          Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg
          465 470 475 480
          Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly
                          485 490 495
          Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala
                      500 505 510
          Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr
                  515 520 525
          Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Glu Gly Lys Ser Ser Ser Gly
              530 535 540
          Ser Gly Ser Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser
          545 550 555 560
          Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr
                          565 570 575
          Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val
                      580 585 590
          Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser
                  595 600 605
          Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu
              610 615 620
          Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr
          625 630 635 640
          His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly
                          645 650 655
          Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp
                      660 665 670
          Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu
                  675 680 685
          Lys Leu
              690
           <![CDATA[ <210> 16]]>
           <![CDATA[ <211> 681]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000182983 - C4LW16 - mature_protein - ]]>
                 IgG1_AAS_G4S_NPQ_G3S3_sthCD40L
           <![CDATA[ <400> 16]]>
          His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
          1 5 10 15
          Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
                      20 25 30
          Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser His Glu Asp Pro
                  35 40 45
          Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
              50 55 60
          Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
          65 70 75 80
          Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
                          85 90 95
          Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
                      100 105 110
          Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
                  115 120 125
          Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
              130 135 140
          Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
          145 150 155 160
          Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
                          165 170 175
          Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
                      180 185 190
          Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
                  195 200 205
          Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly
              210 215 220
          Gly Gly Gly Ser Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala
          225 230 235 240
          Ser Ser Lys Thr Thr Ser Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr
                          245 250 255
          Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr
                      260 265 270
          Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys
                  275 280 285
          Ser Asn Arg Glu Ala Ser Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys
              290 295 300
          Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn
          305 310 315 320
          Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His Leu Gly
                          325 330 335
          Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr
                      340 345 350
          Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu
                  355 360 365
          Leu Lys Leu Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Asn
              370 375 380
          Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr
          385 390 395 400
          Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn
                          405 410 415
          Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly
                      420 425 430
          Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala
                  435 440 445
          Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly
              450 455 460
          Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ser Ala
          465 470 475 480
          Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu
                          485 490 495
          Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val
                      500 505 510
          Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly Gly
                  515 520 525
          Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Asn Pro Gln Ile Ala Ala
              530 535 540
          His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp
          545 550 555 560
          Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Asn Leu Val Thr Leu Glu
                          565 570 575
          Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr
                      580 585 590
          Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro
                  595 600 605
          Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile
              610 615 620
          Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln
          625 630 635 640
          Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser
                          645 650 655
          Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly
                      660 665 670
          Phe Thr Ser Phe Gly Leu Leu Lys Leu
                  675 680
           <![CDATA[ <210> 17]]>
           <![CDATA[ <211> 686]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000182982 - C4LW15 - mature_protein - ]]>
                 IgG1_AAS_G4S2_NPQ_G3S3_sthCD40L
           <![CDATA[ <400> 17]]>
          His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
          1 5 10 15
          Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
                      20 25 30
          Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser His Glu Asp Pro
                  35 40 45
          Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
              50 55 60
          Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
          65 70 75 80
          Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
                          85 90 95
          Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
                      100 105 110
          Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
                  115 120 125
          Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
              130 135 140
          Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
          145 150 155 160
          Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
                          165 170 175
          Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
                      180 185 190
          Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
                  195 200 205
          Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly
              210 215 220
          Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Pro Gln Ile Ala Ala His
          225 230 235 240
          Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala
                          245 250 255
          Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn
                      260 265 270
          Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala
                  275 280 285
          Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe
              290 295 300
          Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu
          305 310 315 320
          Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln
                          325 330 335
          Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val
                      340 345 350
          Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe
                  355 360 365
          Thr Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly Ser Gly Gly Gly Ser
              370 375 380
          Gly Gly Gly Ser Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala
          385 390 395 400
          Ser Ser Lys Thr Thr Ser Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr
                          405 410 415
          Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr
                      420 425 430
          Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys
                  435 440 445
          Ser Asn Arg Glu Ala Ser Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys
              450 455 460
          Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn
          465 470 475 480
          Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His Leu Gly
                          485 490 495
          Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr
                      500 505 510
          Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu
                  515 520 525
          Leu Lys Leu Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Asn
              530 535 540
          Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr
          545 550 555 560
          Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn
                          565 570 575
          Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly
                      580 585 590
          Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala
                  595 600 605
          Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly
              610 615 620
          Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ser Ala
          625 630 635 640
          Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu
                          645 650 655
          Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val
                      660 665 670
          Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu
                  675 680 685
           <![CDATA[ <210> 18]]>
           <![CDATA[ <211> 691]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000182981 - C4LW14 - mature_protein - ]]>
                 IgG1_AAS_G4S3_NPQ_G3S3_sthCD40L
           <![CDATA[ <400> 18]]>
          His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
          1 5 10 15
          Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
                      20 25 30
          Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser His Glu Asp Pro
                  35 40 45
          Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
              50 55 60
          Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
          65 70 75 80
          Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
                          85 90 95
          Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
                      100 105 110
          Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
                  115 120 125
          Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
              130 135 140
          Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
          145 150 155 160
          Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
                          165 170 175
          Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
                      180 185 190
          Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
                  195 200 205
          Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly
              210 215 220
          Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Pro
          225 230 235 240
          Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser
                          245 250 255
          Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu
                      260 265 270
          Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu
                  275 280 285
          Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser
              290 295 300
          Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg
          305 310 315 320
          Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ser Ala Lys
                          325 330 335
          Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln
                      340 345 350
          Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser
                  355 360 365
          His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly
              370 375 380
          Ser Gly Gly Gly Ser Gly Gly Gly Ser Asn Pro Gln Ile Ala Ala His
          385 390 395 400
          Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala
                          405 410 415
          Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn
                      420 425 430
          Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala
                  435 440 445
          Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe
              450 455 460
          Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu
          465 470 475 480
          Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln
                          485 490 495
          Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val
                      500 505 510
          Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe
                  515 520 525
          Thr Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly Ser Gly Gly Gly Ser
              530 535 540
          Gly Gly Gly Ser Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala
          545 550 555 560
          Ser Ser Lys Thr Thr Ser Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr
                          565 570 575
          Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr
                      580 585 590
          Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys
                  595 600 605
          Ser Asn Arg Glu Ala Ser Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys
              610 615 620
          Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn
          625 630 635 640
          Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His Leu Gly
                          645 650 655
          Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr
                      660 665 670
          Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu
                  675 680 685
          Leu Lys Leu
              690
           <![CDATA[ <210> 19]]>
           <![CDATA[ <211> 696]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000182980 - C4LW13 - mature_protein - ]]>
                 IgG1_AAS_G4S4_NPQ_G3S3_sthCD40L
           <![CDATA[ <400> 19]]>
          His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
          1 5 10 15
          Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
                      20 25 30
          Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser His Glu Asp Pro
                  35 40 45
          Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
              50 55 60
          Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
          65 70 75 80
          Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
                          85 90 95
          Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
                      100 105 110
          Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
                  115 120 125
          Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
              130 135 140
          Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
          145 150 155 160
          Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
                          165 170 175
          Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
                      180 185 190
          Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
                  195 200 205
          Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly
              210 215 220
          Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
          225 230 235 240
          Gly Gly Ser Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser
                          245 250 255
          Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr
                      260 265 270
          Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val
                  275 280 285
          Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser
              290 295 300
          Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu
          305 310 315 320
          Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr
                          325 330 335
          His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly
                      340 345 350
          Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp
                  355 360 365
          Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu
              370 375 380
          Lys Leu Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Asn Pro
          385 390 395 400
          Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser
                          405 410 415
          Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu
                      420 425 430
          Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu
                  435 440 445
          Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser
              450 455 460
          Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg
          465 470 475 480
          Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ser Ala Lys
                          485 490 495
          Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln
                      500 505 510
          Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser
                  515 520 525
          His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly
              530 535 540
          Ser Gly Gly Gly Ser Gly Gly Gly Ser Asn Pro Gln Ile Ala Ala His
          545 550 555 560
          Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala
                          565 570 575
          Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn
                      580 585 590
          Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala
                  595 600 605
          Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe
              610 615 620
          Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu
          625 630 635 640
          Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln
                          645 650 655
          Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val
                      660 665 670
          Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe
                  675 680 685
          Thr Ser Phe Gly Leu Leu Lys Leu
              690 695
           <![CDATA[ <210> 20]]>
           <![CDATA[ <211> 143]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> CD40L v.1]]>
           <![CDATA[ <400> 20]]>
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          1 5 10 15
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                      20 25 30
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                  35 40 45
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
              50 55 60
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Trp Leu Lys Ser Pro
          65 70 75 80
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
                          85 90 95
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                      100 105 110
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                  115 120 125
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu
              130 135 140
           <![CDATA[ <210> 21]]>
           <![CDATA[ <211> 141]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> CD40L v.2]]>
           <![CDATA[ <400> 21]]>
          Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser
          1 5 10 15
          Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu
                      20 25 30
          Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu
                  35 40 45
          Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser
              50 55 60
          Ser Gln Ala Pro Phe Ile Ala Ser Leu Trp Leu Lys Ser Pro Gly Arg
          65 70 75 80
          Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ser Ala Lys
                          85 90 95
          Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln
                      100 105 110
          Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser
                  115 120 125
          His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu
              130 135 140
           <![CDATA[ <210> 22]]>
           <![CDATA[ <211> 143]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> CD40L v.3]]>
           <![CDATA[ <400> 22]]>
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          1 5 10 15
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                      20 25 30
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                  35 40 45
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
              50 55 60
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
          65 70 75 80
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
                          85 90 95
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                      100 105 110
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                  115 120 125
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu
              130 135 140
           <![CDATA[ <210> 23]]>
           <![CDATA[ <211> 9]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker]]>
           <![CDATA[ <400> 23]]>
          Glu Gly Lys Ser Ser Gly Ser Gly Ser
          1 5
           <![CDATA[ <210> 24]]>
           <![CDATA[ <211> 15]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker]]>
           <![CDATA[ <400> 24]]>
          Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
          1 5 10 15
           <![CDATA[ <210> 25]]>
           <![CDATA[ <211> 12]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker]]>
           <![CDATA[ <400> 25]]>
          Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser
          1 5 10
           <![CDATA[ <210> 26]]>
           <![CDATA[ <211> 10]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker]]>
           <![CDATA[ <400> 26]]>
          Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
          1 5 10
           <![CDATA[ <210> 27]]>
           <![CDATA[ <211> 20]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker]]>
           <![CDATA[ <400> 27]]>
          Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
          1 5 10 15
          Gly Gly Gly Ser
                      20
           <![CDATA[ <210> 28]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker]]>
           <![CDATA[ <400> 28]]>
          Gly Gly Gly Gly Ser
          1 5
           <![CDATA[ <210> 29]]>
           <![CDATA[ <211> 229]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Fc Monomer v.1]]>
           <![CDATA[ <400> 29]]>
          Val Glu Arg Lys Ser Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro
          1 5 10 15
          Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
                      20 25 30
          Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
                  35 40 45
          Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
              50 55 60
          Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
          65 70 75 80
          Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu
                          85 90 95
          Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala
                      100 105 110
          Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro
                  115 120 125
          Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
              130 135 140
          Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
          145 150 155 160
          Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
                          165 170 175
          Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
                      180 185 190
          Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
                  195 200 205
          Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
              210 215 220
          Leu Ser Pro Gly Lys
          225
           <![CDATA[ <210> 30]]>
           <![CDATA[ <211> 232]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Fc monomer v.2]]>
           <![CDATA[ <400> 30]]>
          Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
          1 5 10 15
          Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
                      20 25 30
          Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
                  35 40 45
          Val Ser Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
              50 55 60
          Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
          65 70 75 80
          Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
                          85 90 95
          Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
                      100 105 110
          Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
                  115 120 125
          Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
              130 135 140
          Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
          145 150 155 160
          Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
                          165 170 175
          Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
                      180 185 190
          Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
                  195 200 205
          Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
              210 215 220
          Ser Leu Ser Leu Ser Pro Gly Lys
          225 230
           <![CDATA[ <210> 31]]>
           <![CDATA[ <211> 223]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Fc monomer v.3]]>
           <![CDATA[ <400> 31]]>
          His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser
          1 5 10 15
          Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
                      20 25 30
          Thr Pro Glu Val Thr Cys Val Val Val Ser Val Ser His Glu Asp Pro
                  35 40 45
          Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
              50 55 60
          Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
          65 70 75 80
          Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
                          85 90 95
          Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
                      100 105 110
          Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
                  115 120 125
          Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
              130 135 140
          Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
          145 150 155 160
          Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
                          165 170 175
          Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
                      180 185 190
          Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
                  195 200 205
          Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
              210 215 220
           <![CDATA[ <210> 32]]>
           <![CDATA[ <211> 669]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> TPP000161222 - C40W33 - mature_protein]]>
           <![CDATA[ <400> 32]]>
          Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val
          1 5 10 15
          Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val
                      20 25 30
          Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr
                  35 40 45
          Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser
              50 55 60
          Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe
          65 70 75 80
          Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro
                          85 90 95
          Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro
                      100 105 110
          Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His
                  115 120 125
          Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Glu Gly Lys Ser
              130 135 140
          Ser Gly Ser Gly Ser Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser
          145 150 155 160
          Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr
                          165 170 175
          Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val
                      180 185 190
          Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser
                  195 200 205
          Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu
              210 215 220
          Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr
          225 230 235 240
          His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly
                          245 250 255
          Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp
                      260 265 270
          Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu
                  275 280 285
          Lys Leu Glu Gly Lys Ser Ser Gly Ser Gly Ser Gln Ile Ala Ala His
              290 295 300
          Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala
          305 310 315 320
          Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn
                          325 330 335
          Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala
                      340 345 350
          Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe
                  355 360 365
          Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu
              370 375 380
          Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln
          385 390 395 400
          Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val
                          405 410 415
          Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe
                      420 425 430
          Thr Ser Phe Gly Leu Leu Lys Leu Val Glu Arg Lys Ser Cys Val Glu
                  435 440 445
          Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu
              450 455 460
          Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
          465 470 475 480
          Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln
                          485 490 495
          Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
                      500 505 510
          Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu
                  515 520 525
          Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
              530 535 540
          Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
          545 550 555 560
          Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
                          565 570 575
          Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
                      580 585 590
          Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
                  595 600 605
          Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly
              610 615 620
          Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
          625 630 635 640
          Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
                          645 650 655
          His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
                      660 665
           <![CDATA[ <210> 33]]>
           <![CDATA[ <211> 140]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> CD40L v.4]]>
           <![CDATA[ <400> 33]]>
          Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val
          1 5 10 15
          Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val
                      20 25 30
          Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr
                  35 40 45
          Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser
              50 55 60
          Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe
          65 70 75 80
          Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro
                          85 90 95
          Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro
                      100 105 110
          Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His
                  115 120 125
          Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu
              130 135 140
           <![CDATA[ <210> 34]]>
           <![CDATA[ <211> 141]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> CD40L v.5]]>
           <![CDATA[ <400> 34]]>
          Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser
          1 5 10 15
          Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu
                      20 25 30
          Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu
                  35 40 45
          Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser
              50 55 60
          Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg
          65 70 75 80
          Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ser Ala Lys
                          85 90 95
          Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln
                      100 105 110
          Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser
                  115 120 125
          His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu
              130 135 140
           <![CDATA[ <210> 35]]>
           <![CDATA[ <211> 443]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>CD40L trimer v.1]]>
           <![CDATA[ <400> 35]]>
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          1 5 10 15
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                      20 25 30
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                  35 40 45
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
              50 55 60
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Trp Leu Lys Ser Pro
          65 70 75 80
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
                          85 90 95
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                      100 105 110
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                  115 120 125
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Glu
              130 135 140
          Gly Lys Ser Ser Gly Ser Gly Ser Gln Ile Ala Ala His Val Ile Ser
          145 150 155 160
          Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
                          165 170 175
          Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln
                      180 185 190
          Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr
                  195 200 205
          Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser
              210 215 220
          Leu Trp Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala
          225 230 235 240
          Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His
                          245 250 255
          Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn
                      260 265 270
          Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
                  275 280 285
          Gly Leu Leu Lys Leu Glu Gly Lys Ser Ser Gly Ser Gly Ser Gln Ile
              290 295 300
          Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu
          305 310 315 320
          Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr
                          325 330 335
          Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr
                      340 345 350
          Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Ser Gln
                  355 360 365
          Ala Pro Phe Ile Ala Ser Leu Trp Leu Lys Ser Pro Gly Arg Phe Glu
              370 375 380
          Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys
          385 390 395 400
          Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly
                          405 410 415
          Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly
                      420 425 430
          Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu
                  435 440
           <![CDATA[ <210> 36]]>
           <![CDATA[ <211> 453]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>CD40L trimer v.2]]>
           <![CDATA[ <400> 36]]>
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          1 5 10 15
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                      20 25 30
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                  35 40 45
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
              50 55 60
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
          65 70 75 80
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
                          85 90 95
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                      100 105 110
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                  115 120 125
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly
              130 135 140
          Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Asn Pro Gln Ile Ala
          145 150 155 160
          Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln
                          165 170 175
          Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Asn Leu Val Thr Leu
                      180 185 190
          Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile
                  195 200 205
          Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Ser Gln Ala
              210 215 220
          Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg
          225 230 235 240
          Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly
                          245 250 255
          Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala
                      260 265 270
          Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr
                  275 280 285
          Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Gly Gly Gly Ser Gly Gly
              290 295 300
          Gly Ser Gly Gly Gly Ser Asn Pro Gln Ile Ala Ala His Val Ile Ser
          305 310 315 320
          Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
                          325 330 335
          Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln
                      340 345 350
          Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr
                  355 360 365
          Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser
              370 375 380
          Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala
          385 390 395 400
          Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His
                          405 410 415
          Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn
                      420 425 430
          Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
                  435 440 445
          Gly Leu Leu Lys Leu
              450
           <![CDATA[ <210> 37]]>
           <![CDATA[ <211> 447]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>CD40L trimer v.3]]>
           <![CDATA[ <400> 37]]>
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          1 5 10 15
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                      20 25 30
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                  35 40 45
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
              50 55 60
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
          65 70 75 80
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
                          85 90 95
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                      100 105 110
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                  115 120 125
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Glu
              130 135 140
          Gly Lys Ser Ser Gly Ser Gly Ser Asn Pro Gln Ile Ala Ala His Val
          145 150 155 160
          Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu
                          165 170 175
          Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly
                      180 185 190
          Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln
                  195 200 205
          Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile
              210 215 220
          Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu
          225 230 235 240
          Arg Ala Ala Asn Thr His Ser Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser
                          245 250 255
          Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe
                      260 265 270
          Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr
                  275 280 285
          Ser Phe Gly Leu Leu Lys Leu Glu Gly Lys Ser Ser Gly Ser Gly Ser
              290 295 300
          Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr
          305 310 315 320
          Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn
                          325 330 335
          Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln
                      340 345 350
          Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu
                  355 360 365
          Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro
              370 375 380
          Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser
          385 390 395 400
          Ala Lys Pro Cys Gly Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu
                          405 410 415
          Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln
                      420 425 430
          Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu
                  435 440 445
           <![CDATA[ <210> 38]]>
           <![CDATA[ <211> 440]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223>CD40L trimer v.4]]>
           <![CDATA[ <400> 38]]>
          Ile Ala Ala His Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val
          1 5 10 15
          Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val
                      20 25 30
          Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr
                  35 40 45
          Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser
              50 55 60
          Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe
          65 70 75 80
          Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro
                          85 90 95
          Cys Gly Gln Gln Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro
                      100 105 110
          Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His
                  115 120 125
          Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu Glu Gly Lys Ser
              130 135 140
          Ser Gly Ser Gly Ser Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser
          145 150 155 160
          Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr
                          165 170 175
          Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val
                      180 185 190
          Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser
                  195 200 205
          Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu
              210 215 220
          Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr
          225 230 235 240
          His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly
                          245 250 255
          Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp
                      260 265 270
          Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu
                  275 280 285
          Lys Leu Glu Gly Lys Ser Ser Gly Ser Gly Ser Gln Ile Ala Ala His
              290 295 300
          Val Ile Ser Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala
          305 310 315 320
          Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn
                          325 330 335
          Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala
                      340 345 350
          Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe
                  355 360 365
          Ile Ala Ser Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu
              370 375 380
          Leu Arg Ala Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln
          385 390 395 400
          Ser Ile His Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val
                          405 410 415
          Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe
                      420 425 430
          Thr Ser Phe Gly Leu Leu Lys Leu
                  435 440
           <![CDATA[ <210> 39]]>
           <![CDATA[ <211> 4]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> MISC_Features]]>
           <![CDATA[ <222> (1)..(4)]]>
           <![CDATA[ <223> GGGS can be repeated n times, where n is an integer from 1 to 20]]>
           <![CDATA[ <400> 39]]>
          Gly Gly Gly Ser
          1               
           <![CDATA[ <210> 40]]>
           <![CDATA[ <211> 5]]>
           <![CDATA[ <212> PRT]]>
           <![CDATA[ <213> Artificial Sequence]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <223> Linker]]>
           <![CDATA[ <220>]]>
           <![CDATA[ <221> MISC_Features]]>
           <![CDATA[ <222> (1)..(5)]]>
           <![CDATA[ <223> GGGS can be repeated n times, where n is an integer from 1 to 20]]>
           <![CDATA[ <400> 40]]>
          Gly Gly Gly Gly Ser
          1 5
          
      

Claims (54)

A polypeptide comprising a single-chain trimeric CD40 ligand (CD40L) fusion protein, wherein the single-chain trimeric CD40L fusion protein comprises three CD40L subunits covalently linked to each other by a peptide linker ( CD40L trimer), Optionally wherein the CD40L subunits comprise a portion of the CD40L extracellular domain. The polypeptide according to claim 1, wherein the CD40L subunits comprise any one of the sequences selected from SEQ ID NO: 20 to 22, or a fragment thereof. The polypeptide of claim 1 or 2, wherein at least one of the peptide linkers is selected from the group consisting of EGKSSGSGS (SEQ ID NO:23) and (G ₃ S) ₃ (SEQ ID NO:25) ; optionally wherein at least two of the peptide linkers have the same sequence. The polypeptide according to any one of claims 1 to 3, wherein the single-chain trimeric CD40L fusion protein comprises any sequence selected from SEQ ID NO: 35 to 38, or a fragment thereof. The polypeptide according to any one of claims 1 to 4, wherein the single-chain trimeric CD40L fusion protein is fused to a peptide or polypeptide not derived from CD40L. The polypeptide according to any one of claims 1 to 5, wherein the single-chain trimeric CD40L fusion protein is fused to a peptide tether; optionally wherein the peptide tether is selected from (G ₄ S) ₃ (SEQ ID NO:24), (G ₄ S) ₂ (SEQ ID NO:26), (G ₄ S) ₄ (SEQ ID NO:27), and G ₄ S (SEQ ID NO:28) and/or optionally wherein the peptide tether is fused to the N-terminus of the single-chain trimeric CD40L fusion protein or the C-terminus of the single-chain trimeric CD40L fusion protein. The polypeptide according to any one of claims 1 to 6, wherein the single-chain trimeric CD40L fusion protein is fused to an Fc monomer peptide. The polypeptide of claim 7, wherein the Fc monomer peptide comprises a human Fc sequence; Optionally wherein the human Fc sequence comprises a sequence selected from the group consisting of immunoglobulins IgG, IgA, IgM, IgD, and IgE, Optionally, wherein the IgG sequence is selected from IgG1, IgG2, IgG3, and IgG4, optionally among (i) the IgG sequence comprises an IgG1 sequence, and the IgG1 sequence comprises SEQ ID NO: 30 or 31, or a fragment thereof, or (ii) the IgG sequence comprises an IgG2 sequence, and the IgG2 sequence comprises SEQ ID NO: 29, or a fragment thereof. The polypeptide of claim 7 or 8, wherein the single-chain trimeric CD40L fusion protein is fused to the Fc monomer peptide via a peptide tether; optionally wherein the peptide tether comprises 0 to 20 amino acids; may Optionally wherein the peptide tether is selected from (G ₄ S) ₃ (SEQ ID NO: 24), (G ₄ S) ₂ (SEQ ID NO: 26), (G ₄ S) ₄ (SEQ ID NO: 27), and the group consisting of G ₄ S (SEQ ID NO: 28). The polypeptide according to any one of claims 7 to 9, wherein the CD40L trimer system is linked to: (a) the N-terminus of the Fc monomer peptide, Optionally, wherein the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 1 to 12, or a fragment thereof; or (b) the C-terminus of the Fc monomer peptide, Optionally, wherein the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 13 to 19, or a fragment thereof. The polypeptide according to any one of claims 1 to 10, wherein the single-chain trimeric CD40L fusion protein: (a) enhance the activation of CD40 polypeptide compared to wild-type CD40L; Optionally, wherein the activation of the CD40 polypeptide comprises (i) Enhanced immunostimulatory function of T cells and/or B cells; (ii) B cells, CD4+ T cells, CD8+ T cells, dendritic cells, macrophages, natural killer cells, monocytes, granulocytes, eosinophils, and/or spheroids compared to wild-type CD40L Enhanced activation of neutrophils; and/or (iii) increased expression of the CD40 polypeptide. (b) Enhanced anti-tumor activity compared to wild-type CD40L; (c) enhanced pro-inflammatory activity compared to wild-type CD40L; (d) Enhanced clearance of infectious pathogens compared to wild-type CD40L; (e) increase the antibody production of B cell population by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70% , about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%; (f) Increase the secretion of pro-inflammatory cytokines in the T cell population; Optionally, wherein the pro-inflammatory cytokines are IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN- γ, or any combination thereof; Optionally, wherein the cytokine production is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70% %, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%; (g) increasing the minimum percentage of phagocytic macrophages in the macrophage population to about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%; and/or (h) increasing the minimum percentage of antigen-presenting dendritic cells in the population of dendritic cells to about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60% , about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. The polypeptide according to any one of claims 1 to 11, wherein the polypeptide is conjugated to a medicament; Optionally wherein the agent is a radioisotope, metal chelator, enzyme, fluorescent compound, bioluminescent compound, or chemiluminescent compound. A single chain trimeric CD40L Fc fusion protein comprising: (a) three CD40L subunits covalently linked to each other by a peptide linker (CD40L trimer); and (b) an Fc monomeric peptide. The single-chain trimeric CD40L Fc fusion protein according to claim 13, wherein the peptide linker is EGKSSGSGS (SEQ ID NO: 23) or (G ₃ S) ₃ (SEQ ID NO: 25). The single-chain trimeric CD40L Fc fusion protein as claimed in claim 113 or 14, wherein the Fc monomer peptide is covalently linked to the CD40L trimer by a peptide tether, optionally wherein the peptide tether comprises Between 0 and 20 amino acids, and/or optionally wherein the peptide tether is selected from (G ₄ S) ₃ (SEQ ID NO:24), (G ₄ S) ₂ (SEQ ID NO :26), (G ₄ S) ₄ (SEQ ID NO: 27), and the group consisting of G ₄ S (SEQ ID NO: 28). The single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 15, wherein the CD40 ligand subunits comprise a part of the CD40L extracellular domain. The single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 16, wherein the CD40L trimer system is linked to (a) the N-terminus of the Fc monomer peptide, Optionally, wherein the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 1 to 12, or a fragment thereof; or (b) the C-terminus of the Fc monomer peptide, Optionally, wherein the single-chain trimeric CD40L Fc fusion protein comprises any one sequence selected from SEQ ID NO: 13 to 19, or a fragment thereof. The single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 17, wherein the CD40 ligand subunits comprise any one of the sequences selected from SEQ ID NO: 20 to 22, or a fragment thereof. The single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 18, wherein the Fc monomer peptide comprises a human Fc sequence, Optionally wherein the human Fc sequence comprises a sequence selected from the group consisting of immunoglobulins IgG, IgA, IgM, IgD, and IgE, Optionally, wherein the IgG sequence is selected from IgG1, IgG2, IgG3, and IgG4, optionally among (i) the IgG sequence comprises an IgG1 sequence, and the IgG1 sequence comprises SEQ ID NO: 30 or 31, or a fragment thereof, or (ii) the IgG sequence comprises an IgG2 sequence, and the IgG2 sequence comprises SEQ ID NO: 29, or a fragment thereof. The single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 19, wherein the single-chain trimeric CD40L Fc fusion protein: (a) enhance the activation of CD40 polypeptide compared to wild-type CD40L; Optionally, wherein the activation of the CD40 polypeptide comprises (i) Enhanced immunostimulatory function of T cells and/or B cells; (ii) B cells, CD4+ T cells, CD8+ T cells, dendritic cells, macrophages, natural killer cells, monocytes, granulocytes, eosinophils, and/or spheroids compared to wild-type CD40L Enhanced activation of neutrophils; and/or (iii) increased expression of the CD40 polypeptide. (b) Enhanced anti-tumor activity compared to wild-type CD40L; (c) enhanced pro-inflammatory activity compared to wild-type CD40L; (d) Enhanced clearance of infectious pathogens compared to wild-type CD40L; (e) increase the antibody production of B cell population by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70% , about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%; (f) Increase the secretion of pro-inflammatory cytokines in the T cell population; Optionally, wherein the pro-inflammatory cytokines are IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN- γ, or any combination thereof; Optionally, wherein the cytokine production is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70% %, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%; (g) increasing the minimum percentage of phagocytic macrophages in the macrophage population to about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%; and/or (h) increasing the minimum percentage of antigen-presenting dendritic cells in the population of dendritic cells to about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60% , about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. A dimer comprising two single-chain trimeric CD40L Fc fusion proteins according to any one of claims 13 to 20; optionally among (i) the dimer is the same dimer; or (ii) The dimerization system is formed by the association of the Fc monomeric peptides. A polynucleotide encoding the polypeptide according to any one of claims 1 to 12, the single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 20, or the dimer according to claim 21. A vector comprising the polynucleotide according to claim 22. A host cell comprising the polynucleotide according to claim 22 or the vector according to claim 23. A pharmaceutical composition comprising: (i) a pharmaceutically acceptable carrier; and (ii) The polypeptide according to any one of claims 1 to 12, the single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 20, or the dimer according to claim 21. A set comprising the polypeptide according to any one of claims 1 to 12, the single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 20, or the dimer according to claim 21. A method for generating: (a) a single-chain trimeric CD40L Fc fusion protein or a fragment thereof, the method comprising: (i) introducing a polynucleotide into the host cell, the polynucleotide encoding the single-stranded trimeric CD40L Fc fusion protein according to any one of claims 8 to 15; (ii) cultivating the host cell under conditions to produce the single-chain trimeric CD40L Fc fusion protein or a fragment thereof; and (iii) recovering the single-chain trimeric CD40L Fc fusion protein or fragment thereof from the cell or culture; or (b) a dimer comprising two single-chain trimeric CD40L Fc fusion proteins, the method comprising: (i) introducing a polynucleotide into the host cell, the polynucleotide encoding the single-stranded trimeric CD40L Fc fusion protein according to any one of claims 8 to 15; (ii) cultivating the host cell under conditions to produce the single-chain trimeric CD40L Fc fusion protein or fragment thereof; (iii) recovering the single-chain trimeric CD40L Fc fusion protein or fragment thereof from the cell or culture; and (iv) combining a single-chain trimeric CD40L Fc fusion protein or fragment thereof under conditions favorable for dimerization; or (c) A pharmaceutical composition of a single-chain trimeric CD40L Fc fusion protein or a fragment thereof, the method comprising combining the single-chain trimeric CD40L Fc fusion protein or a fragment thereof according to any one of claims 8 to 15 with a pharmaceutically acceptable combination of carriers to obtain the pharmaceutical composition. A system comprising components for providing the following: the polypeptide according to any one of claims 1 to 12, the single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 20, or the polypeptide according to claim 21 of dimers. A method of activating the following: (a) CD40 polypeptide, the method comprises: (i) contacting the CD40 polypeptide with a polypeptide according to any one of claims 1 to 12, wherein the single-chain trimeric CD40L fusion protein activates the CD40 polypeptide upon binding; (ii) contacting the CD40 polypeptide with a single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 20, wherein the single-chain trimeric CD40L Fc fusion protein activates the CD40 polypeptide upon binding; or (iii) contacting the CD40 polypeptide with a dimer comprising two single-chain trimeric CD40L Fc fusion proteins according to any one of claims 13 to 20, wherein the single-chain trimeric CD40L Fc fusion protein The dimer activates the CD40 polypeptide upon binding; (b) target cells, the method comprising: (i) contacting the target cell with the polypeptide according to any one of claims 1 to 12, wherein the single-chain trimeric CD40L fusion protein activates the target cell upon binding; (ii) contacting the target cell with the single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 20, wherein the single-chain trimeric CD40L Fc fusion protein activates the target cell upon binding; or (iii) contacting the cell with a dimer comprising two single-chain trimeric CD40L Fc fusion proteins according to any one of claims 13 to 20, wherein the single-chain trimeric CD40L Fc fusion protein two The aggregate activates the target cell upon binding; Optionally, wherein the target cell line is an antigen-presenting cell; Optionally, the target cell line is B cells, dendritic cells, macrophages, monocytes, granules, or eosinophils, or a combination thereof; Optionally, wherein the target cell is a B cell; Optionally, wherein the target cell is a dendritic cell; Optionally, wherein the target cell is a macrophage; Optionally wherein upon activation, the target cell activates a second cell; optionally wherein the second cell is a T cell, a neutrophil, or a combination thereof; optionally wherein the second cell is a CD4+ T cells, CD8+ T cells, mucosa-associated invariant T (mucosal associated invariant T, MAIT) cells, natural killer cells, neutrophils, or a combination thereof. The method of claim 29, wherein the method is a method of activating a target cell, wherein the activation of the target cell is measured as: (a) increased proliferation or maturation of the target cell; Optionally, wherein the proliferation or maturation of the target cell is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, About 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300% %, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%; or (b) prolonging the survival time of the target cell; Optionally wherein the survival time of the target cells is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300% , about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%. The method according to claim 29 or 30, wherein the contacting further comprises administering a pharmaceutical composition, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and the single-chain trimeric CD40L Fc fusion protein; Optionally wherein the contacting enhances an innate anti-neoplastic immune response. A method of treating cancer in a subject comprising administering to the subject: (a) a therapeutically effective amount of the polypeptide according to any one of claims 1 to 12; (b) a therapeutically effective amount of the single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 20; or (c) a therapeutically effective amount of a dimer comprising two single-chain trimeric CD40L Fc fusion proteins according to any one of claims 13 to 20. The method according to claim 32, which further comprises administering a pharmaceutical composition, the pharmaceutical composition comprising a pharmaceutically acceptable carrier and the single-chain trimeric CD40L Fc fusion protein, Optionally wherein the treatment enhances an innate anti-tumor immune response. The method of claim 32 or 33, further comprising co-administration of a second therapy. The method of any one of claims 32 to 34, wherein the cancer is selected from the group consisting of melanoma, mesothelioma, advanced solid tumors, and lymphoma. A method for promoting antibody production in a population of B cells comprising (a) contacting the population of B cells with a polypeptide according to any one of claims 1 to 12, wherein the single-chain trimeric CD40L fusion protein activates the B cells upon binding; (b) contacting the population of B cells with a single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 20, wherein the single-chain trimeric CD40L Fc fusion protein activates the B cells upon binding; or (c) contacting the population of B cells with a dimer comprising two single-chain trimeric CD40L Fc fusion proteins according to any one of claims 13 to 20, wherein the single-chain trimeric CD40L Fc is fused The protein dimer activates the B cells upon binding; Optionally wherein the antibody production of the B cell population is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, About 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300% %, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%. The method of claim 36, wherein the B cell population is contacted in the presence of an antigen, and wherein the antibody produced by the B cells specifically binds to the antigen; Optionally wherein the method further promotes the formation of memory B cells capable of producing the antibody in response to the antigen. A method of increasing antigen presentation of a population of dendritic cells, comprising contacting the dendritic cells with an effective amount of the following in the presence of the antigen: (i) the polypeptide according to any one of claims 1 to 12; (ii ) the single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 20; or (iii) a dimer comprising two single-chain trimeric CD40L Fc according to any one of claims 13 to 20 fusion protein; Optionally, wherein the minimum percentage of dendritic cells presenting the antigen in the population of dendritic cells is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55% , about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. The method of claim 38, wherein the antigen presentation of the dendritic cells is measured by co-culturing the dendritic cells labeled with the first fluorescent dye with the antigen labeled with the second fluorescent dye, wherein the first fluorescent dye is different from the second fluorescent dye; Optionally the percentage of dendritic cells in which the antigen is presented is measured by determining the percentage of dendritic cells in the population of dendritic cells that co-localize with the antigen. A method of increasing secretion of pro-inflammatory cytokines in an immune cell population, comprising: (a) contacting the population of immune cells with a population of antigen-presenting cells in the presence of a polypeptide according to any one of claims 1 to 12, wherein the single-chain trimeric CD40L fusion protein activates the antigen-presenting cells upon binding; (b) contacting the population of immune cells with a population of antigen-presenting cells in the presence of a single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 20, wherein the single-chain trimeric CD40L Fc fusion protein upon binding activate the antigen-presenting cells; or (c) contacting the population of immune cells with a population of antigen-presenting cells in the presence of a dimer comprising two single-chain trimeric CD40L Fc fusion proteins according to any one of claims 13 to 20, wherein the The single chain trimeric CD40L Fc fusion protein dimer activates the antigen presenting cells upon binding; wherein upon activation, the antigen-presenting cells activate the population of immune cells; optionally wherein the antigen presenting cells present antigen to the population of immune cells; Optionally, wherein the cytokine is IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN-γ, or any combination thereof; Optionally, wherein the cytokine production is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70% %, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000%; Optionally wherein, upon activation of the antigen presenting cells, the antigen presentation to the population of immune cells is increased; Optionally, wherein the minimum percentage of antigen presenting cells presenting the antigen in the population of antigen presenting cells is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55% , about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%; Optionally, wherein the antigen-presenting cells comprise dendritic cells, macrophages, B cells, or a combination thereof; optionally, wherein the immune cell population comprises T cells, neutrophils, or a combination thereof. The method according to any one of claims 36 to 40, wherein the polypeptide, the single-chain trimeric CD40L Fc fusion protein, or the dimer system is in a vaccine composition or an adjuvant composition. The method according to any one of claims 36 to 41, wherein the antigen is in a vaccine composition. The method of any one of claims 36 to 42, wherein the antigen is derived from or derived from: (a) infection with pathogens; Optionally, wherein the infection pathogenic system virus, bacteria, fungi, parasites, or a combination thereof; (b) diseased cells; (c) cells infected with an infectious pathogen; Optionally, wherein the infection pathogenic system virus, bacteria, fungus, parasite, or a combination thereof; or (d) Cancer cells. The method according to any one of claims 36 to 43, wherein the antigen is presented by an antigen-presenting cell; Optionally wherein the antigen presenting cell line is dendritic cells; Optionally wherein the antigen is associated with an MHC class I complex or an MHC class II complex. A method of increasing the phagocytosis of diseased cells by a population of macrophages, comprising contacting the diseased cells, the macrophages, or both the diseased cells and the macrophages with an effective amount of: (i ) a polypeptide according to any one of claims 1 to 12; (ii) a single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 20; or (iii) a dimer comprising two such The single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 20; Optionally, wherein the minimum percentage of phagocytic macrophages in the population of macrophages is increased by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%; Optionally wherein the phagocytosis of macrophages is measured by co-cultivating macrophages labeled with a first fluorescent dye with diseased cells labeled with a second fluorescent dye, wherein the first fluorescent dye The dye is different from the second fluorescent dye. The method of claim 45, wherein the percentage of phagocytic macrophages is measured by determining the percentage of macrophages comprising the diseased cells; Optionally, wherein the diseased cells are cancer cells or cells infected with infectious pathogens; Optionally, the infection pathogenic system is virus, bacteria, fungus, parasite, or a combination thereof. A method for increasing the expression of a CD40 polypeptide in a target cell, comprising contacting the target cell with an effective amount of: (i) the polypeptide according to any one of claims 1 to 12; (ii) any one of claims 13 to 20 Any single-chain trimeric CD40L Fc fusion protein of any one; or (iii) a dimer comprising two single-chain trimeric CD40L Fc fusion proteins according to any one of claims 13-20. The method of claim 47, wherein the target cell line: (a) diseased cells; (b) cancer cells; (c) cells infected with an infectious pathogen; Optionally, wherein the infection pathogenic system virus, bacteria, fungus, parasite, or a combination thereof; or (d) B cells, natural killer cells, dendritic cells, macrophages, monocytes, granulocytes, eosinophils, neutrophils, or combinations thereof; Optionally wherein the diseased cell population is reduced by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70% , about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. A method of creating a pro-inflammatory environment in tissue surrounding a diseased cell population, comprising contacting the tissue with an effective amount of: (i) the polypeptide according to any one of claims 1 to 12; (ii) the polypeptide according to claim 13 The single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 20; or (iii) a dimer comprising two single-chain trimeric CD40L Fc fusion proteins according to any one of claims 13 to 20; Optionally where: (a) Activated B cells, CD4+ T cells, CD8+ T cells, dendritic cells, macrophages, natural killer cells, monocytes, granulocytes, eosinophils, and/or neutrophils in the tissue increased infiltration; (b) the concentration of pro-inflammatory cytokines is increased in the tissue; Optionally, wherein the pro-inflammatory cytokines are IL-1, IL-2, IL-6, IL-12, IL-17, IL-22, IL-23, GM-CSF, TNF-α, IFN- γ, or any combination thereof; (c) antigen presentation by antigen-presenting cells derived from or derived from those diseased cells is increased in the tissue; (d) the phagocytosis of the diseased cells is increased in the tissue; (e) Apoptosis of the diseased cells caused by cell-mediated cytotoxicity is increased in the tissue; (f) Apoptosis of the diseased cells caused by antibody-dependent cellular cytotoxicity is increased in the tissue; and/or (g) the diseased cell population is reduced in the tissue; Optionally, wherein the diseased cell population is reduced by about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% in the tissue , about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. A method for eliminating diseased cells in a subject, comprising administering to the subject an effective amount of: (i) the polypeptide according to any one of claims 1 to 12; (ii) the polypeptide according to any one of claims 13 to 20 A single-chain trimeric CD40L Fc fusion protein; or (iii) a dimer comprising two single-chain trimeric CD40L Fc fusion proteins as claimed in any one of claims 13 to 20; Optionally where: (a) The diseased cells do not express CD40 polypeptide; (b) The diseased cells express CD40 polypeptide; (c) the diseased cell is a cancer cell; or (d) The diseased cell line is a cell infected by an infectious pathogen; Optionally, the infection pathogenic system is virus, bacteria, fungus, parasite, or a combination thereof. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of: (i) the polypeptide according to any one of claims 1 to 12; (ii) any one of claims 13 to 20 The single-chain trimeric CD40L Fc fusion protein of claim 13; or (iii) a dimer comprising two single-chain trimeric CD40L Fc fusion proteins as claimed in any one of claims 13 to 20; optionally among (a) The treatment enhances the innate, humoral, or cell-mediated anti-tumor immune response; (b) the method further comprises co-administration of a second therapy; and/or (c) The cancer is selected from the group consisting of melanoma, mesothelioma, advanced solid tumors, and lymphoma. A method of treating an infection in a subject in need thereof, comprising administering to the subject an effective amount of: (i) the polypeptide according to any one of claims 1 to 12; (ii) any one of claims 13 to 20 The single-chain trimeric CD40L Fc fusion protein of claim 13; or (iii) a dimer comprising two single-chain trimeric CD40L Fc fusion proteins as claimed in any one of claims 13 to 20; Optionally where: (a) The treatment enhances the innate, humoral, or cell-mediated immune response against infection; (b) the subject is co-administered with a vaccine composition for the prophylaxis of the infection in the subject; Optionally wherein the vaccine compositions are co-administered simultaneously or sequentially. A method for increasing the response of a subject in need to an antigen, comprising administering to the subject an effective amount of: (i) the polypeptide according to any one of claims 1 to 12; (ii) as claimed in items 13 to 20 Any single-chain trimeric CD40L Fc fusion protein of any one; or (iii) a dimer comprising two single-chain trimeric CD40L Fc fusion proteins according to any one of claims 13 to 20; Optionally wherein the antigen is an antigen of a cancer, tumor, pathogen, or allergen. A method for increasing the response of a subject in need to a vaccine, comprising administering to the subject the vaccine and an effective amount of: (i) the polypeptide according to any one of claim items 1 to 12; (ii) claim item 13 The single-chain trimeric CD40L Fc fusion protein according to any one of claims 13 to 20; or (iii) a dimer comprising two single-chain trimeric CD40L Fc fusion proteins according to any one of claims 13 to 20; Optionally wherein the vaccine is a vaccine against a tumor, cancer, pathogen, or allergen.

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